Isolated peptides derived from the B7 ligand dimer interface and uses thereof

ABSTRACT

Disclosed are peptides comprising amino acid residues of the dimer interface of human B7-1 and B7-2 and compositions and uses thereof.

The Sequence Listing in ASCII text file format of 22,919 bytes in size,created on Jan. 21, 2020, with the file name“2020-01-21SequenceListing_KAEMPFER20B,” filed in the U.S. Patent andTrademark Office on Jan. 21, 2020, is hereby incorporated herein byreference.

TECHNOLOGICAL FIELD

Disclosed are peptides derived from the dimer interface of the B7ligands. Specifically, the disclosure pertains to novel isolated,non-naturally occurring peptides, compositions comprising thereof andmethods of treatment employing the same. Specific compositions andmethods relate to treatment of infections induced by Gram positive andGram negative bacteria and bacterial components thereof.

BACKGROUND ART

References considered to be relevant as background to the presentlydisclosed subject matter are listed below:

-   [1] Schwartz, J. C., Zhang, X., Fedorov, A. A., Nathenson, S. G.,    and Almo, S. C. (2001). Structural basis for co-stimulation by the    human CTLA-4/B7-2 complex. Nature 410, 604-608.-   [2] Sharpe, A. H., and Freeman, G. J. (2002). The B7-CD28    superfamily. Nat. Rev. Immunol. 2, 116-126.-   [3] Riley, J. L., and June, C. H. (2005). The CD28 family: a T-cell    rheostat for therapeutic control of T-cell activation. Blood 105,    13-21.-   [4] Collins, A. V., Brodie, D. W., Gilbert, R. J., laboni, A.,    Manso-Sancho, R., Walse, B., Stuart, D. I., van der Merwe, P. A.,    and Davis, S. J. (2002). The interaction properties of costimulatory    molecules revisited. Immunity 17, 201-210.-   [5] Greenwald, R. J., Freeman, G. J., and Sharpe, A. H. (2005). The    B7 family revisited. Annu. Rev. Immunol. 23, 515-548.-   [6] Bhatia, S., Edidin, M., Almo, S. C., and Nathenson, S. G.    (2006). B7-1 and B7-2: similar costimulatory ligands with different    biochemical, oligomeric and signaling properties. Immunol. Lett.    104, 70-75.-   [7] Marrack, P., Blackman, M., Kushnir, E., and Kappler, J. (1990).    The toxicity of staphylococcal enterotoxin B in mice is mediated by    T cells. J. Exp. Med. 171, 455-464.-   [8] Miethke, T., Wahl, C., Heeg, K., Echtenacher, B., Krammer, P.    H., and Wagner, H. (1992). T cell-mediated lethal shock triggered in    mice by the superantigen staphylococcal enterotoxin B: critical role    of tumor necrosis factor. J. Exp. Med. 175, 91-98.-   [9] Leder, L. et al. (1998). A mutational analysis of the binding of    staphylococcal enterotoxins B and C3 to the T cell receptor beta    chain and major histocompatibility complex class II. J. Exp. Med.    187, 823-833.-   [10] Arad, G., Levy, R., Nasie, I., Hillman, D., Rotfogel, Z.,    Barash, U., Supper, E., Shpilka, T., Minis, A., and Kaempfer, R.    (2011). Binding of superantigen toxins into the CD28 homodimer    interface is essential for induction of cytokine genes that mediate    lethal shock. PLoS Biol. 9, e1001149.-   [11] WO 2004/087196.-   [12] Arad, G., Levy, R., Hillman, D., and Kaempfer, R. (2000).    Superantigen antagonist protects against lethal shock and defines a    new domain for T-cell activation. Nat. Med. 6, 414-421.-   [13] Ramachandran, G. et al. (2013). A peptide antagonist of CD28    signaling attenuates toxic shock and necrotizing soft-tissue    infection induced by Streptococcus pyogenes. J. Infect. Dis. 207,    1869-1877.-   [14] Guerrier-Takada, C., Eder, P. S., Gopalan, V., and Altman, S.    (2002). Purification and characterization of Rpp25, an RNA-binding    protein subunit of human ribonuclease P. RNA 8, 290-295.

These publications are referred to below by their above numbers.Acknowledgement of the above references herein is not to be inferred asmeaning that these are in any way relevant to the patentability of thepresently disclosed subject matter.

BACKGROUND

As a principal co-stimulatory receptor, CD28 is a critical regulator ofthe immune response (1-3). Expressed constitutively on T cells, CD28 isa homodimer that interacts with its B7 coligands, namely, B7-1 (CD80)and B7-2 (CD86), expressed on antigen-presenting cells. CD28/B7interaction results in transducing the signal essential for an immediateT-cell response (2-4). CD28 coligand B7-2 (CD86) is expressedconstitutively on antigen-presenting cells whereas B7-1 (CD80) isinduced only later (4); hence, B7-2/CD28 interaction regulates earlyantigen signaling (5, 6).

B7-1 and B7-2 are glycoproteins, each consisting of single V-like andC-like immunoglobulin superfamily (IgSF) domains. Their ligands, CD28and CTLA-4, are also structurally related and expressed at the cellsurface as disulfide-linked homodimers of single V-like IgSF domains.

The inflammatory cytokine response is indispensable for protectiveimmunity yet bacterial and viral infections often elicit an exaggeratedresponse (‘cytokine storm’) which is harmful to the host. It is knownthat bacterial superantigens from Staphylococcus aureus andStreptococcus pyogenes induce toxic shock by activating an immuneresponse, orders of magnitude beyond that elicited by regular antigens.It has been previously shown that superantigens exploit the main axis ofT-cell activation by binding directly as intact proteins to most majorhistocompatibility class II (MHC-II) and T-cell receptor (TCR) moleculesoutside their antigen-binding domains, linking them and bypassingrestricted presentation of conventional antigens which typicallyactivate <1% of T cells, thereby activating up to 20-30% of T cells(7-9).

Moreover, T-cell activation by superantigens requires their directbinding to CD28 (10), the second signaling molecule mandatory for T-cellactivation. This results in massive induction of inflammatory cytokinesthat mediate toxic shock, including interleukin-2 (IL2), interferon-γ(IFN-γ) and tumor necrosis factor (TNF).

There are ample examples for the harmful effects of superantigens. Forexample, the majority of human food poisoning cases, manifested byvomiting and diarrhea after ingestion, are caused by the enterotoxins(SEs) family of superantigens, secreted by S. aureus. Among the majorserological types within the SEs family are SEA, SEB, SEE and SEG, whereSEB has been also recognized as a leading cause of human cases ofnon-menstrual toxic shock syndrome that can accompany surgical orinjurious wound infections, as well as viral infections of therespiratory tract. Notably, toxic shock syndrome, in its most severeform, causes shock and death.

Bacterial superantigens are thus known to induce a Th1 cytokine storm,causing toxic shock. The inventors have previously shown that mice wereprotected from lethal superantigen challenge by peptide mimetics of theCD28 dimer interface and by peptides selected to compete with thesuperantigen for its binding site in CD28 (10, 11).

Specifically, induction of human inflammatory cytokine gene expressionby divergent superantigens may be inhibited by a short peptide thatshows homology to a 12-amino-acid β-strand-hinge-α-helix superantigendomain, which is structurally conserved in superantigens yet remote fromtheir MHC-II and TCR binding sites. Through this domain, essential forsuperantigen action (10, 12), superantigens engage CD28 directly at itshomodimer interface (10). Blocking access of a superantigen to CD28,with peptide mimetics of the CD28 homodimer interface or theβ-strand-hinge-α-helix superantigen domain, suffices to block signalingfor overexpression of inflammatory cytokines in human peripheral bloodmononuclear cells (PBMC) and to protect mice from lethal toxic shock(10, 12, 13).

GENERAL DESCRIPTION

The present disclosure provides an isolated and purified peptidecomprising at least one amino acid residue of the crystallographic dimerinterface within a region of the extracellular domain of human B7-2,said region consisting of the amino acid sequence denoted by SEQ IDNO:13, wherein said crystallographic dimer interface consists of aminoacid residues Thr-11, Leu-26, Ser-27, Leu-46, Gly-47, Lys-48, Glu-49,Phe-51, Met-59, Gly-60, Arg-61, Thr-62, Ser-63, Phe-64, Asp-65, Ser-66,Asp-67, Arg-72, His-74 and Asn-75 of SEQ ID NO:13, wherein said isolatedand purified peptide further comprises at least 2 additional amino acidresidues at its C-terminus and/or N-terminus, wherein said additionalamino acid residues are consecutive amino acid residues of SEQ ID NO:13immediately adjacent to said at least one amino acid residue of saidcrystallographic dimer interface in SEQ ID NO: 13, wherein said isolatedand purified peptide consists of from 3 to about 30 amino acid residues,and functional fragments and derivatives thereof. These peptides arealso referred to herein as “hB7-2 mimetic peptides”.

In some embodiments the isolated and purified peptide according to thepresent disclosure comprises said at least one amino acid residue of thecrystallographic dimer interface within a region of the extracellulardomain of human B7-2 and from 2 to about 8 said additional amino acidresidues at its C-terminus and/or N-terminus, and functional fragmentsand derivatives thereof.

In other embodiments the isolated and purified peptide according to thepresent disclosure consists of an amino acid sequence selected from theamino acid sequences denoted by SEQ ID NO:11 (MGRTSFDSDS, alsodesignated pB2-7), SEQ ID NO:5 (EKFDSVHSKYM, also designated peptidepB2-4) and SEQ ID NO:9 (DSDSWTLR also designated peptide pB2-6) andfunctional fragments and derivatives thereof.

By a further aspect the present disclosure provides an isolated andpurified peptide comprising at least one amino acid residue of thecrystallographic dimer interface within a region of the extracellulardomain of human B7-1, said region consisting of the amino acid sequencedenoted by SEQ ID NO: 14, wherein said crystallographic dimer interfaceconsists of amino acid residues Val-15, Leu-29, Ala-30, Ser-48, Gly-49,Asp-50, Met-51, Lys-58, Asn-59, Arg-60, Thr-61, Ile-62, Phe-63, Asp-64,Ile-65, Thr-66, Val-72, Leu-74 and Ala-75 of SEQ ID NO:14, wherein saidisolated and purified peptide further comprises at least 2 additionalamino acid residues at its C-terminus and/or N-terminus, wherein saidadditional amino acid residues are consecutive amino acid residues ofSEQ ID NO:14 immediately adjacent to said at least one amino acidresidue of said crystallographic dimer interface in SEQ ID NO: 14,wherein said isolated and purified peptide consists of from 3 to about30 amino acid residues, and functional fragments and derivativesthereof. These peptides are also referred to herein as “hB7-1 mimeticpeptides”.

In some embodiments the isolated and purified peptide according to thepresent disclosure comprises said at least one amino acid residue of thecrystallographic dimer interface within a region of the extracellulardomain of human B7-1 and from 2 to about 8 said additional amino acidresidues at each of its C-terminus and/or N-terminus, and functionalfragments and derivatives thereof.

In other embodiments the isolated and purified peptide according to thepresent disclosure consists of an amino acid sequence selected from theamino acid sequences denoted by SEQ ID NO:38 (MNIWPEYK, designatedpB1-4), SEQ ID NO:40 (KNRTIFDITN, designated pB1-7), SEQ ID NO:42(DITNNLSIV, designated pB1-6), SEQ ID NO:46(SGDMNIWPEYKNRTIFDITNNLSIVILA) and SEQ ID NO:48 (YKNRTIFD, designatedpB1-8) and functional derivatives thereof.

In further embodiments the functional derivative of the presentlydisclosed isolated and purified peptide is any one of:

-   -   i. said B7-2 mimetic peptide that is extended at the N terminus        and/or the C terminus thereof by 1 to 4 consecutive amino acid        residues present in immediately adjacent corresponding positions        of the amino acid sequence denoted by SEQ ID NO: 13;    -   ii. said B7-2 mimetic peptide that is extended at the N terminus        and/or the C terminus thereof:        -   (a) by cysteine or by lauryl cysteine;        -   (b) by an organic moiety that is not naturally occurring or            by a synthetic amino acid residue;        -   (c) by N-acetyl or lysyl-palmitoyl residue;        -   (d) by hydrophobic amino acid residue(s) which may be            naturally occurring or synthetic amino acid residues; or    -   iii. a dimer or multimer of any of the peptides of (i) and (ii);    -   iv. a constrained conformation of said B7-2 mimetic peptide;    -   v. any of said B7-2 mimetic peptides and their derivatives as        defined in (i) to (iv), that is modified by at least one        synthetic mutation selected from insertion, deletion,        substitution, provided that the modified peptide comprises at        least one amino acid residue of the said dimer interface;    -   wherein said derivative consists of from 3 to about 40 amino        acid residues.

In still further embodiments the functional derivative of the presentlydisclosed isolated and purified peptide is any one of:

-   -   i. said B7-1 mimetic peptide that is extended at the N terminus        and/or the C terminus thereof by 1 to 4 consecutive amino acid        residues present in immediately adjacent corresponding positions        of the amino acid sequence denoted by SEQ ID NO: 14;    -   ii. said B7-1 mimetic peptide that is extended at the N terminus        and/or the C terminus thereof:        -   (a) by cysteine or by lauryl cysteine;        -   (b) by an organic moiety that is not naturally occurring or            by a synthetic amino acid residue;        -   (c) by N-acetyl or lysyl-palmitoyl residue;        -   (d) by hydrophobic amino acid residue(s) which may be            naturally occurring or synthetic amino acid residues; or    -   iii. a dimer or multimer of any of the peptides of (i) and (ii);    -   iv. a constrained conformation of said B7-1 mimetic peptide;    -   v. any of said B7-1 mimetic peptides and their derivatives as        defined in (i) to (iv), that is modified by at least one        synthetic mutation selected from insertion, deletion,        substitution, provided that the modified peptide comprises at        least one amino acid residue of the said dimer interface;    -   wherein said derivative consists of from 3 to about 40 amino        acid residues.

In some aspects and embodiments the isolated and purified peptide asherein disclosed is extended at its N terminus and/or at its C terminusby a D-Ala amino acid residue.

In specific embodiments the isolated peptide according to the presentdisclosure is any one of a peptides consisting of the amino acidsequence (D-A)EKFDSVHSKYM(D-A) as denoted by SEQ ID NO:6 (alsodesignated herein as peptide D-Ala-pB2-4), a peptide consisting of theamino acid sequence (D-A)DSDSWTLR(D-A) as denoted by SEQ ID NO: 10 (alsodesignated herein as peptide D-Ala-pB2-6) and a peptide consisting ofthe amino acid sequence (D-A)MGRTSFDSDS(D-A) as denoted by SEQ ID NO: 12(also designated herein as peptide D-Ala-pB2-7).

In other specific embodiments the isolated and purified peptideaccording to the present disclosure is any one of a peptide consistingof the amino acid sequence (D-A)MNIWPEYK(D-A) as denoted by SEQ ID NO:39 (also designated D-Ala-pB1-4), a peptide consisting of the amino acidsequence (D-A)KNRTIFDITN(D-A) as denoted by SEQ ID NO:41 (alsodesignated D-Ala-pB1-7), a peptide consisting of the amino acid sequence(D-Ala)DITNNLSIV(D-Ala) as denoted by SEQ ID NO:43 (also designatedD-Ala-pB1-6), and a peptide consisting of the amino acid sequence(D-Ala)YKNRTIFD(D-Ala) denoted by SEQ ID NO:49 (also designatedD-Ala-pB1-8).

The present disclosure further provides a pharmaceutical compositioncomprising as an active ingredient at least one isolated and purifiedpeptide as herein defined, optionally further comprising apharmaceutically acceptable carrier, diluent, adjuvant and/or excipient.

In some embodiments the pharmaceutical composition according to thepresent disclosure is for eliciting protective immunity against at leastone of sepsis, toxic shock, septic shock, severe sepsis, incapacitationand resulting death, that are induced by a bacterial pathogen, a mixtureof bacterial pathogens and/or a toxic bacterial component.

In other embodiments the pharmaceutical composition according to thepresent disclosure is for the treatment of at least one of a bacterialinfection and acute inflammation associated therewith in a humansubject.

In all aspects and embodiments the at least one of bacterial infectionand acute inflammation associated therewith is induced by at least oneof Gram-positive bacteria, Gram-negative bacteria and toxic bacterialcomponents.

In all aspects and embodiments the Gram-negative bacteria are selectedfrom the group consisting of proteobacteria, Escherichia coli,Salmonella, Shigella, Enterobacteriaceae, Pseudomonas, Moraxella,Helicobacter, Bdellovibrio, Stenotrophomonas, acetic acid bacteria,Legionella, alpha-proteobacteria, Wolbachia, Gram-negative cocci,Neisseria species, Neisseria gonorrhoeae, Neisseria meningitidis,Moraxella catarrhalis, Gram-negative bacilli, Hemophilus influenzae,Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa,Proteus mirabilis, Enterobacter cloacae, Serratia marcescens,Helicobacter pylori, Salmonella enteritidis, Salmonella typhi,Acinetobacter baumannii, Francisella tularemia, Vibrio, vulnificus,cholerae, fluvialis, parahemolyticus, alginolyticus, Photobacterdamsela, Aeromonas hydrophila, Clostridium perfringens, Clostridiumhistolyticum, Porphyromonas/prevotella sp. Prevotella Intermedia,Prevotella Buccae, Prevotella sp., Bacteroides uniformis and NDM-1bacterial strains, the Gram-positive bacteria are selected from thegroup consisting of Group A streptococcus, S. pyogenes, S. pneumonia,Group B strep, Enterococcus faecalis, Group D streptococcus, Group Gstreptococcus, Streptococcus viridans, Streptococcus milleri,Propionibacterium sp., Enterococcus faecium, Peptostreptococcus sp.,Streptococcus Microaerophilic, Lactobacillus sp., StaphylococcusEpidermis and Staphylococcus aureus, and the toxic bacterial componentsare selected from the group consisting of exotoxins, endotoxins,superantigenic toxins, pathogen associated molecular patterns (PAMPs),Damage Associated Molecular Pattern molecules (DAMPs),lipopolysaccharides, peptidoglycans or toxic components thereof,molecules that are associated with groups of pathogens that arerecognized by cells of the innate immune system and molecules that areassociated with groups of pathogens that are recognized by Toll-likereceptors (TLRs).

In some embodiments the at least one toxic bacterial component is asuperantigenic toxin.

In other embodiments the pharmaceutical composition according to thepresent disclosure is for any one of oral administration and parenteraladministration.

In all aspects and embodiments the parenteral administration as hereindefined is any one of intravenous, intramuscular, intraperitoneal,intranasal, intrathecal subcutaneous injection or said administration isby inhalation.

The present disclosure further provides a peptide as herein defined foruse in a method for eliciting in a human subject in need protectiveimmunity against at least one of sepsis, toxic shock, septic shock,severe sepsis, incapacitation and resulting death, that are induced by abacterial pathogen, a mixture of bacterial pathogens and/or a toxicbacterial component.

By another one of its aspects the present disclosure provides a peptideas herein defined for use in a method of treating at least one ofbacterial infection and acute inflammation associated therewith in ahuman subject in need.

The present disclosure further provides a pharmaceutical composition asherein defined for use in a method for eliciting in a human subject inneed protective immunity against at least one of sepsis, toxic shock,septic shock, severe sepsis, incapacitation and resulting death, thatare induced by a bacterial pathogen, a mixture of bacterial pathogensand/or a toxic bacterial component.

By still another one of its aspects the presently disclosed subjectmatter provides a pharmaceutical composition comprising as an activeingredient at least one isolated and purified peptide as herein definedfor use in a method of treating at least one of bacterial infection andacute inflammation associated therewith in a human subject in need,wherein said pharmaceutical composition optionally further comprises apharmaceutically acceptable carrier, diluent, adjuvant and/or excipient.

The presently disclosed subject matter further provides a method foreliciting in a human subject in need protective immunity against atleast one of sepsis, toxic shock, septic shock, severe sepsis,incapacitation and resulting death, that are induced by a bacterialpathogen, a mixture of bacterial pathogens and/or at least one toxicbacterial component, said method comprising administering to saidsubject an immunologically effective amount of a peptide as hereindefined or of a pharmaceutically acceptable composition comprising thesame.

In some embodiments the method of treating at least one of bacterialinfection and acute inflammation associated therewith further comprisesadministering an additional therapeutic agent to said human subject inneed.

In some embodiments the immunologically effective amount is from about0.1 to about 60 μg/Kg body weight of said subject.

The present disclosure further provides a method of treating at leastone of bacterial infection and acute inflammation associated therewithin a human subject in need, comprising administering to said subject atherapeutically effective amount of at least one peptide or acomposition comprising the same as herein defined.

In some embodiments the therapeutically effective amount as hereindefined is from about 0.1 to about 60 μg/Kg body weight of said subject.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosedherein and to exemplify how it may be carried out in practice,embodiments will now be described, by way of non-limiting example only,with reference to the accompanying drawings, in which:

FIG. 1 : Peptides Derives from the Homodimer Interface of B7-2.

The amino acid sequence of human B7-2 (hB7-2) dimer interface fragment((1), in which the N-terminal amino acid residue proline (P) is replacedby methionine (M), as denoted by SEQ ID NO: 50), on which the hB7-2peptide mimetics are denoted. In the extracellular domain of hB7-2,residues in the dimer interface are underlined and residues that contactCTLA4 are in boldface. Peptide sequences are framed.

FIG. 2A-FIG. 2E: Peptide Mimetics of the B7-2 Homodimer Interface areSuperantigen Antagonists.

FIG. 2A1-FIG. 2A5 show diagrams of the level of IFN-γ in the presence ofthe indicated peptides. PBMC were induced with SEB (10 ng/ml), in theabsence (open circles in each panel) or presence of 0.1 μg/ml peptidepB2-2 (FIG. 2A1), pB2-3 (FIG. 2A2), pB2-4 (FIG. 2A3), pB2-5 (FIG. 2A4)and pB2-6 (FIG. 2A5) (filled circles), as shown. Secreted IFN-γ wasdetermined (pg/ml×10⁻²) (means±SEM; n=3).

FIG. 2B1-FIG. 2B10 show diagrams of the levels of IL2 in the presence ofthe indicated peptides, namely pB2-2 (FIG. 2B1), pB2-3 (FIG. 2B2), pB2-4(FIG. 2B3), pB2-5 (FIG. 2B4) and pB2-6 (FIG. 2B5), and TNF-α in thepresence of peptides pB2-2 (FIG. 2B6), pB2-3 (FIG. 2B7), pB2-4 (FIG.2B8), pB2-5 (FIG. 2B9) and pB2-6 (FIG. 2B10). PBMC were activated by SEB(10 ng/ml) in the absence (open circles) or presence of 0.1 μg/ml of theindicated peptides (filled circles). Secreted IL2 and TNF-α weredetermined (error bars, SEM; n=3).

FIG. 2C1-FIG. 2C4 show diagrams of the levels of IL-2 (FIG. 2C1), IFN-γ(FIG. 2C2), TNF-α (FIG. 2C3) and IL-10 (FIG. 2C4) in the presence of thebelow indicated peptides.

PBMC were induced with recombinant SEB (0.1 ng/ml), in the absence (opencircles) or presence of 0.01 μg/ml pB2-4 (grey filled circles), pB2-6(black filled circles) or both (black filled squares). Secretedcytokines were determined (means±SEM; n=3).

FIG. 2D1-FIG. 2D4 show diagrams of the levels of IL-2 (FIG. 2D1), IFN-γ(FIG. 2D2), TNF-α (FIG. 2D3) and IL-10 (FIG. 2D4) in the presence of thebelow-indicated peptides. PBMC were induced with SEB (0.1 ng/ml) alone(open circles) or together with 0.1 g/ml of pB2-7 (filled circles) or0.01 μg/ml of each pB2-4 and pB2-6 (filled squares). Secreted IL-2,IFN-γ, TNF-α and IL-10 were determined (means±SEM; n=3).

FIG. 2E1-FIG. 2E7 show diagrams of the levels of IL-2 (FIG. 2E1 and FIG.2E5), IFN-γ (FIG. 2E2 and FIG. 2E6), TNF-α (FIG. 2E3 and FIG. 2E7) andIL-10 (FIG. 2E4) in the presence of the indicated peptides and SMEZ orTSST-1. PBMC were incubated with 0.01 ng/ml SMEZ (FIG. 2E1, FIG. 2E2,FIG. 2E3 and FIG. 2E4) or TSST-1 (FIG. 2E5, FIG. 2E6 and FIG. 2E7) alone(open circles) or together with 0.1 μg/ml of pB2-7 (filled circles) or0.01 μg/ml of each pB2-4 and pB2-6 (filled squares). Secreted cytokineswere determined (error bars, SEM; n=3).

FIG. 3A-FIG. 3F: Peptide Mimetics of the B7-2 Dimer Interface BindSuperantigens and Block Binding of Superantigens to Cell-Surface B7-2and CD28.

FIG. 3A-FIG. 3B show representative SPR responses diagrams for bindingof SEB in twofold increments from 0.78 μM to immobilized pB2-4 (FIG. 3A)and pB2-6 (FIG. 3B).

FIG. 3C-FIG. 3F show representative SPR responses diagrams for bindingof TSST-1 in five twofold increments from 0.0625 μM (FIG. 3C and FIG.3D) and of SMEZ in four twofold increments from 0.0625 μM (FIG. 3E andFIG. 3F) to the immobilized peptides pB2-4 (FIG. 3C and FIG. 3E) orpB2-6 (FIG. 3D and FIG. 3F), using the chips of FIG. 3A-3B.

FIG. 4A-FIG. 4D: The B7-2 Peptide Mimetics Inhibit Binding of SEB toB7-2 or CD28.

FIG. 4A-FIG. 4C show representative Western blots of inhibition ofbinding of SEB to cell-surface B7-2 by peptide mimetics of the B7-2 orCD28 homodimer interface. HEK-293T cells were transfected to expressB7-2 or with empty vector (EV) and after 36 hours were incubated for 1hour without addition (B7-2) or as indicated, with 5 μg/ml cB7-2antibody, αCD28 monoclonal antibody (10) or 10 μg/ml of B7-2 mimeticpeptides pB2-2, pB2-4, pB2-6 or pB2-7, or CD28 mimetic peptides p1TA orp2TA (10) before further incubation for 1 hour with 15 μg/ml recombinantSEB. Cells were washed 3 times with cold phosphate-buffered salinebefore lysis. Western blots of equal amounts of total cell protein(Bradford assay) with 0.1 μg/ml αSEB antibody followed by 0.2 μg/mlhorseradish peroxidase-conjugated donkey anti-mouse IgG (top) or with0.1 μg/ml cB7-2 antibody followed by 0.2 μg/ml horseradishperoxidase-conjugated donkey anti-goat IgG (bottom).

FIG. 4D shows a representative Western blot of inhibition of binding ofSEB to cell-surface CD28 by peptide mimetics of the B7-2 or CD28homodimer interface. HEK-293T cells were transfected to express CD28(10) or with empty vector before incubation with peptides and SEB asabove. Western blots show binding of SEB to CD28 (10) (top) andexpression of CD28, assayed with 0.1 μg/ml αCD28 antibody (bottom).

FIG. 5A-FIG. 5B: Peptide Mimetics of the B7-2 Dimer Interface ProtectMice from Lethal SEB Challenge.

FIG. 5A is a diagram showing survival of mice (n=5 per group) injectedwith SEB (10 μg) alone (open circles) or together with 1 μg of each ofthe peptide pB2-4 (filled squares) or pB2-6 (filled triangles); p forsurvival, 0.022.

FIG. 5B is a diagram showing survival of mice (n=6 per group) injectedwith SEB alone (open circles) or together with 0.2 μg of the peptidepB2-7 (filled circles); p for survival, 0.005. Peptides wereadministered 30 min before injection of SEB.

FIG. 6A-FIG. 6B: B7-2 Dimer Interface Mimetic Peptide pB2-7 does notInhibit Signaling Through the T Cell Receptor.

Human PBMC were induced with αCD3 alone (open triangles), or with αCD3in the presence of 1 or 10 μg/ml of pB2-7 (filled triangles) (FIG. 6Aand FIG. 6B, respectively). Human PBMC were also induced in the presenceof 1 or 10 μg/ml of pB2-7 alone (FIG. 6A and FIG. 6B, respectively,filled circles). At the indicated time points, IFN-γ secreted into theculture medium was determined by ELISA.

FIG. 7A-FIG. 7B: B7-2 Dimer Interface Mimetic Peptide pB2-7 AttenuatesSignaling Through CD28.

Human PBMC were induced with αCD3/αCD28 alone (open circles), or withαCD3/αCD28 in the presence of 0.01 μg/ml pB2-7 (filled triangles) or0.001 μg/ml pB2-7 (filled circles). At the indicated time points, IFN-γ(FIG. 7A) and TNF-α (FIG. 7B) secreted into the culture medium weredetermined by ELISA.

FIG. 8A-FIG. 8B: B7-2 Dimer Interface Mimetic Peptide pB2-4 AttenuatesSignaling Through CD28.

Human PBMC were induced with αCD3/αCD28 alone (open circles), or withαCD3/αCD28 in the presence of 0.1 μg/ml of pB2-4 (filled squares) or inthe presence of 1 μg/ml of pB2-2 (filled triangles). At the indicatedtime points, IFN-γ (FIG. 8A) and TNF-α (FIG. 8B) secreted into theculture medium were determined by ELISA.

FIG. 9A-FIG. 9B: B7-2 Dimer Interface Mimetic Peptides AttenuateLPS-Mediated Induction of TNF-α.

Human PBMC were induced with E. coli LPS at 25 ng/ml alone (opencircles), or with LPS in the presence of 0.1 μg/ml of pB2-4 (filleddiamonds), pB2-6 (filled triangles), or both (filled squares) (FIG. 9A),or with E. coli LPS at 0.1 μg/ml alone (open circles), or with LPS inthe presence of 0.1 μg/ml of pB2-7 (filled circles) (FIG. 9B). At theindicated time points, TNF-α secreted into the culture medium wasdetermined by ELISA.

FIG. 10 : B7-2 Dimer Interface Mimetic Peptides Attenuate LPS-MediatedInduction of TNF-α.

Human PBMC were induced with E. coli LPS alone (open circles), or withLPS in the presence of 0.01 μg/ml of the peptides pB2-4 (filledtriangles), pB2-6 (filled circles), or pB2-7 (filled squares). At theindicated time points, TNF-α secreted into the culture medium wasdetermined by ELISA.

FIG. 11 : Peptides Derives from the Homodimer Interface of B7-1.

The amino acid sequence of human B7-1 (hB7-1) dimer interface fragmenton which the hB7-1 peptide mimetics are denoted (SEQ ID NO:57).Conserved residues between hB7-2 and hB7-1 are indicated in boldface.Peptide sequences are framed.

FIG. 12 : Sequence alignment of segments of the dimer interface of hB7-2and hB7-1. The sequence alignment shows the amino acid sequence of thepeptides pB2-4, pB2-7 and pB2-6, derived from the dimer interface ofhB7-2 (SEQ ID NO:58) and the corresponding amino acid sequences of thepeptides derived from the dimer interface of hB7-1 (SEQ ID NO:57),namely pB1-4, pB1-7 and pB1-6. Identical amino acids are shown in boldface.

DETAILED DESCRIPTION OF EMBODIMENTS

The presently disclosed subject matter is based on the surprisingfinding that superantigens bind not only directly to CD28, as previouslyshown, but also to the CD28 coligand, namely, to B7-2. It is shownherein that binding of B7-2 by the superantigen is essential forsuperantigen function.

It is also shown herein that superantigens engage B7-2 at its homodimerinterface. As evident from the Examples presented below, short peptidemimetics of the B7-2 dimer interface bind diverse superantigens, inhibitsuperantigen-mediated induction of IL2, IFN-γ and TNF-α in humanperipheral blood mononuclear cells, and are effective antagonists invivo, protecting mice from lethal superantigen challenge.

The B7-2 dimer interface thus serves as a novel therapeutic target forsuperantigen intoxication. The present findings provide a novel,host-oriented therapeutic approach to the sequelae of superantigenintoxication involving blocking of the indispensible interaction of asuperantigen with the dimer interface of B7-2, or with the dimerinterface of CD28, through peptides that mimic the dimer interface ofB7-2.

Thus far, B7-2 was only considered to function as costimulatory ligandin the immune response. The Examples presented below reveal anunexpected and novel role for B7-2 as a receptor for a class ofmicrobial pathogens, the superantigens. Thus, through direct binding,superantigens make unconventional use not only of CD28 (10), MHC-II andTCR, but also of B7-2.

Short peptide mimetics of the B7-2 homodimer interface are shown hereinto be capable of inhibiting the superantigen-mediated induction of IL2,IFN-γ and TNF-α in human peripheral blood mononuclear cells, asdemonstrated, for instance, in Example 1. Moreover, these short peptidemimetics of the B7-2 homodimer interface are also shown to be effectiveSEB antagonists in vivo, protecting mice from lethal toxin challenge, asdemonstrated in Example 3 and FIG. 5 .

The present disclosure also shows that superantigens engage B7-2directly at its dimer interface, as implied from the interactionobserved for SEB and peptides derived from the dimer interface of B7-2(FIG. 3A and FIG. 3B).

The dimer interface of B7-2 thus serves as a novel therapeutic targetfor superantigen intoxication. In particular, for example, peptidesD-Ala-pB2-4 and D-Ala-pB2-6 (abutted at both ends by D-Ala, as denotedby SEQ ID NO: 6 and SEQ ID NO: 10, respectively) which are derived fromthe dimer interface of B7-2 as shown in FIG. 1 , blocked the lethalityof a specific superantigen, SEB, in mice. As shown in FIG. 5 , whileonly ⅕ control mice survived SEB challenge, survival was demonstratedfor ⅘ and 5/5 mice that received D-Ala-pB2-4 and D-Ala-pB2-6,respectively, at the time of SEB administration. Remarkably, thepeptides were protective when present in only 2- to 2.4-fold molarexcess over SEB. Furthermore, D-Ala-pB2-7 (denoted by SEQ ID NO: 12)which is also derived from the dimer interface of B7-2 as shown in FIG.1 , and spans a region of the dimer interface of B7-2 that overlaps withboth the C-terminal region of pB2-4 and the N-terminal region of pB2-6,was shown to be protective against SEB challenge in mice, with 6/6 micesurviving, even when D-Ala-pB2-7 (denoted by SEQ ID NO:12) was presentin near-equal molar ratio to SEB (FIG. 5 ). This high efficacy suggeststhat the B7-2 dimer interface plays a critical role in mediating thedeleterious response to superantigens.

The finding that not only CD28 but also its coligand B7-2 is a directsensor of a class of microbial pathogens, the superantigens, broadensthe scope of pathogen pattern recognition mechanisms.

A direct consequence of the findings presented herein below, is thatpeptide mimetics of either B7-2 or CD28 dimer interface possess dualantagonist activity, interfering in a reciprocal manner with binding ofthe superantigen to either receptor. This dual action, documented here,may explain why D-Ala-pB2-7 (having an amino acid sequence denoted bySEQ ID NO:12), a decapeptide mimetic of the B7-2 dimer interface, orp2TA, an octapeptide mimetic of the CD28 dimer interface (10), caneffectively protect mice from lethal challenge with SEB, despite thatfact that the superantigen, a 238-amino acid protein molecule, interactswith the TCR and MHC-II molecule in addition to binding either B7-2 orCD28.

It was previously reported that the superantigen engages the TCR andMHC-II molecule as well as CD28 with micromolar affinity (10),consistent with the results shown herein below, that B7-2 dimerinterface peptides also exhibit micromolar affinity for diversesuperantigens (see Table 2). The moderate affinity of a superantigen forits four receptors, including B7-2, can explain why peptides bindingwith a similarly moderate affinity can disrupt the synapse and thusattenuate the Th1 cytokine response.

It was also previously reported that activation of a Th1 cytokineresponse by SEB depends upon B7-2 but not B7-1 and is abrogated by amonoclonal antibody directed against B7-2 (10). While these resultscould be interpreted by a need for costimulation through the interactionof B7-2 with CD28 in the MYPPPY (SEQ ID NO:59) domain, the inventors'findings presented below demonstrate that the superantigen engages B7-2directly at a distinct domain, the composite dimer interface (1). Thatthis binding is critical for superantigen action is strongly supportedby the potent superantigen antagonist activity of B7-2 dimer interfacepeptides, as shown below, both ex vivo and in vivo.

Although CD28 and B7-2 differ in amino acid sequence and structure attheir homodimer interfaces and do not form heterodimers, thesuperantigen is capable of binding to either of these receptors with anaffinity in the micromolar range.

Formation of a quaternary complex between superantigen and MHC-II, TCRand either CD28 or B7-2 is structurally feasible. Within theimmunological synapse between antigen-presenting cell and T cell, it isassumed that multiple CD28 and B7-2 molecules engage each other asB7-2/CD28 pairs, thereby creating a network (1). Without being bound bytheory, it is thus conceivable that binding of the superantigen to onlyone receptor within a given pair, rather than to both, may suffice for Tcell hyperactivation, as long as both receptors are engaged within theimmunological synapse. In the synapse, multiple superantigen moleculesmust cooperatively engage CD28 and B7-2 to achieve signaling for harmfulinflammatory cytokine induction, though not for induction of IL10. Thesefindings modify the current view of superantigen action.

The B7-2 dimer interface has no known role in costimulation and is wellseparated from the CD28 binding site, as previously reported. Engagementof a superantigen should displace contacts between the B7-2 monomers,which unlike CD28 are not linked through an intermolecular disulfidebond outside the dimer interface and thus require a second-order bindingreaction to re-dimerize. The Examples shown below support theinterpretation that the superantigen induces a conformational change inB7-2 that activates signaling, likely through the B7-2/CD28 axis. SEBinduces vigorous expression of Th1 and Th2 cytokines but only the Th1response, here defined by induction of the IL2, IFN-γ and TNF-α genes,depends on B7-2 engagement. This mirrors the selective requirement forCD28 in the induction of IL2, IFN-γ and TNF-α genes by superantigens,yet not for the induction of IL4 and IL10 (10). By attenuating thisresponse, the antagonist peptides according to the present disclosurewill reduce the synergy between these cytokines, to allow survival.

Thus, based on the inventors' present findings, provided herewith is anovel host-oriented therapeutic approach to the sequelae of superantigenintoxication involving blocking of the indispensible interaction of asuperantigen with the dimer interface of B7-2, through peptides thatmimic the dimer interface of B7-2.

Therefore, presently disclosed is an isolated and purified peptidecomprising at least one amino acid residue of the crystallographic dimerinterface within a region of the extracellular domain of human B7-2,said region consisting of the amino acid sequence denoted by SEQ IDNO:13, wherein said crystallographic dimer interface consists of aminoacid residues Thr-11, Leu-26, Ser-27, Leu-46, Gly-47, Lys-48, Glu-49,Phe-51, Met-59, Gly-60, Arg-61, Thr-62, Ser-63, Phe-64, Asp-65, Ser-66,Asp-67, Arg-72, His-74 and Asn-75 of SEQ ID NO:13, wherein said isolatedand purified peptide further comprises at least 2 additional amino acidresidues at its C-terminus and/or N-terminus, wherein said additionalamino acid residues are consecutive amino acid residues of SEQ ID NO:13immediately adjacent to said at least one amino acid residue of saidcrystallographic dimer interface in SEQ ID NO: 13, wherein said isolatedand purified peptide consists of from 3 to about 30 amino acid residues,and functional fragments and derivatives thereof.

Human B7-1 (CD80, Accession number NM_005191) and B7-2 (CD86, Accessionnumber 1185_A) as referred to herein are as described in (1). Unlessindicated differently, the terms “B7-1” and “B7-2” refer to human B7-1(also referred to as “hB7-1”) and human B7-2 (also referred to as“hB7-2”), respectively.

As used herein, the term “crystallographic dimer interface” refers tothe contact points formed between two identical monomeric proteinmolecules. Without being bound by theory, these contact points are basedon hydrophobic bonding, van der Waals forces, and salt bridges formedbetween specific amino acid residues comprised in the interface of eachof the monomeric protein molecules, thereby forming a dimerizationinterface. As known in the art and as shown herein below, the specificamino acid residues comprised in the interface of each of the monomericprotein molecules and engaged in forming the dimer interface are notnecessarily positioned in the primary amino acid sequence as consecutiveamino acid residues. The terms “crystallographic dimer interface ofB7-1” and “dimer interface of B7-1” are used herein interchangingly. Theterms “crystallographic dimer interface of B7-2” and “dimer interface ofB7-2” are used herein interchangingly.

In particular, the presently disclosed subject matter relates to thedimer interface formed between monomeric hB7-2 protein molecules (alsoreferred to as the crystallographic dimer interface of hB7-2). Specificresidues that participate in forming the human B7-2 dimer interface werepreviously reported by Schwartz et al. (1) and are underlined in FIG. 1. The dimer interface of B7-2 is located within a region of theextracellular domain of human B7-2 and thus the presently disclosedsubject matter provides isolated and purified peptides consisting of atleast one amino acid residue of the dimer interface within a region ofthe extracellular domain of human B7-2.

Therefore, as detailed above, the presently disclosed subject matterprovides an isolated and purified peptide comprising at least one aminoacid residue of the dimer interface within a region of the extracellulardomain of human B7-2, wherein the region of the extracellular domain ofhuman B7-2 consists of the amino acid sequence denoted by SEQ ID NO:13,namely, the amino acid sequencePLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELS VLA. Thefollowing amino acid residues participate in the formation of the dimerinterface between monomeric B7-2 protein molecules: Thr-11, Leu-26,Ser-27, Leu-46, Gly-47, Lys-48, Glu-49, Phe-51, Met-59, Gly-60, Arg-61,Thr-62, Ser-63, Phe-64, Asp-65, Ser-66, Asp-67, Arg-72, His-74 andAsn-75 based on the numbering of SEQ ID NO:13.

The isolated and purified peptide according to this disclosure thusconsists of at least one of the amino acid residues participating in theformation of the dimer interface between monomeric B7-2 proteinmolecules recited above, and at least two additional amino acid residuesat each of its C-terminus and/or N-terminus, wherein said additionalamino acid residues are consecutive amino acid residues of SEQ ID NO: 13immediately adjacent to said at least one amino acid residue of saiddimer interface in SEQ ID NO: 13, wherein said isolated and purifiedpeptide consists of from 3 to about 30 amino acid residues, andfunctional fragments and derivatives thereof.

Thus, in the above and other embodiments, the length of the isolated andpurified peptide according to the present disclosure is from 3 to about30 amino acid residues. In some embodiments the length of the isolatedand purified peptide according to the present disclosure is from 3 to29, from 3 to 28, from 3 to 27, from 3 to 26, from 3 to 25, from 3 to24, from 3 to 23, from 3 to 22, from 3 to 21, from 3 to 20, from 3 to19, from 3 to 18, from 3 to 17, from 3 to 16, from 3 to 15, from 3 to14, from 3 to 13, from 3 to 12, from 3 to 11, from 3 to 10, from 3 to 9,from 3 to 8, from 3 to 7, from 3 to 6, from 3 to 5, from 3 to 4 aminoacid residues. Specific peptides consist of 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13 and 14 amino acid residues. Derivatives of the peptides accordingto the present invention wherein the peptide is extended by one or moreamino acid residues can be longer, as described below

In the above and other embodiments of the presently disclosed subjectmatter the isolated and purified peptide according to the inventioncomprises said at least one amino acid residue of said crystallographicdimer interface and from 2 to about 8, for example 2 to 8, 3 to 8, 4 to8, 5 to 8, 6 to 8 or 7 to 8, more specifically 2, 3, 4, 5, 6, 7 or 8said additional amino acid residues at its C-terminus and/or N-terminus,and functional fragments and derivatives thereof.

The term “consecutive amino acid residues of an amino acid sequence” asherein defined refers to amino acid residues that correspond to aminoacid residues of a reference amino acid sequence (namely SEQ ID NO: 13or SEQ ID NO: 14) in positions that are immediately adjacent to said atleast one amino acid residue of said dimer interface and which arecontiguous (located one after the other) in the reference amino acidsequence.

By the term “immediately adjacent to an amino acid in an amino acidsequence” as used herein is meant an amino acid residue next to, orimmediately preceding, or immediately following this amino acid residuein the corresponding position/s in the reference amino acid residues.

In some embodiments, the isolated and purified peptide of the presentlydisclosed subject matter, consists of an amino acid sequence selectedfrom the amino acid sequences denoted by SEQ ID NO:5 (EKFDSVHSKYM, alsodesignated peptide pB2-4), SEQ ID NO:9 (DSDSWTLR also designated peptidepB2-6), SEQ ID NO:11 (MGRTSFDSDS, also designated pB2-7), SEQ ID NO:18(FNETADLP), SEQ ID NO:20 (NQSLSELV), SEQ ID NO:22 (YLGKEKFD), SEQ IDNO:24 (TLRLHNLQ), SEQ ID NO:26 (YMGRTSFDSD), and SEQ ID NO:44(LGKEKFDSVHSKYMGRTSFDSDSWTLRLHN), and functional fragments andderivatives thereof.

In other embodiments the isolated and purified peptide according to thepresently disclosed subject matter consists of an amino acid sequenceselected from the amino acid sequences denoted by SEQ ID NO:11(MGRTSFDSDS, also designated pB2-7), SEQ ID NO:5 (EKFDSVHSKYM, alsodesignated peptide pB2-4) and SEQ ID NO:9 (DSDSWTLR also designatedpeptide pB2-6) and functional fragments and derivatives thereof.

In specific embodiments the isolated and purified peptide according tothe presently disclosed subject matter is of the amino acid sequencedenoted by SEQ ID NO:5 (EKFDSVHSKYM, also designated peptide pB2-4).

In other specific embodiments the isolated and purified peptideaccording to the presently disclosed subject matter is of the amino acidsequence denoted by SEQ ID NO:9 (DSDSWTLR also designated peptidepB2-6).

In further specific embodiments the isolated and purified peptideaccording to the presently disclosed subject matter is of the amino acidsequence denoted by SEQ ID NO:11 (MGRTSFDSDS, also designated pB2-7).

It is noteworthy that only isolated and purified peptides comprising atleast one amino acid residue of the dimer interface of the extracellulardomain of human B7-2, as defined above, were shown to actively inhibitcytokine induction. As shown for example in FIG. 2A, the peptidesD-Ala-pB2-2, D-Ala-pB2-3 and D-Ala-pB2-5 (having amino acid sequences asdenoted in Table 3 below) which do not comprise any amino acid residueof said dimer interface, failed to inhibit the SEB-mediated induction ofIFN-γ in human PBMC, whereas D-Ala-pB2-4 and D-Ala-pB2-6 exhibited verystrong inhibitory activity. Likewise, as shown in FIG. 2B, D-Ala-pB2-4and D-Ala-pB2-6, but not the other three peptides D-Ala-pB2-2,D-Ala-pB2-3 and D-Ala-pB2-5 peptides, inhibited the induction of IL2 andTNF-α by SEB.

According to another aspect, the presently disclosed subject matterprovides an isolated and purified peptide comprising at least one aminoacid residue of the crystallographic dimer interface within a region ofthe extracellular domain of human B7-1, said region consisting of theamino acid sequence denoted by SEQ ID NO:14, wherein saidcrystallographic dimer interface consists of amino acid residues Val-15,Leu-29, Ala-30, Ser-48, Gly-49, Asp-50, Met-51, Lys-58, Asn-59, Arg-60,Thr-61, Ile-62, Phe-63, Asp-64, Ile-65, Thr-66, Val-72, Leu-74 andAla-75 of SEQ ID NO: 14, wherein said isolated and purified peptidefurther comprises at least 2 additional amino acid residues at itsC-terminus and/or N-terminus, wherein said additional amino acidresidues are consecutive amino acid residues of SEQ ID NO: 14immediately adjacent to said at least one amino acid residue of saidcrystallographic dimer interface in SEQ ID NO:14, wherein said isolatedand purified peptide consists of from 3 to about 30 amino acid residues,and functional fragments and derivatives thereof.

Thus, as indicated above, the presently disclosed subject matter alsorelates to the dimer interface formed between monomeric proteinmolecules of hB7-1, which is homologous to hB7-2.

B7-1 is homologous to B7-2, both acting as costimulatory ligandsexpressed on the surface of antigen presenting cells (APCs). Binding ofthese molecules to the T cell costimulatory receptors, CD28 and CTLA-4,is essential for the activation and regulation of T cell immunity.Despite strong structural similarities, B7-1 and B7-2 exhibit differentbiochemical features, and their binding to the costimulatory receptorsresults in distinct T cell functional outcomes.

Specific residues that participate in forming the human B7-1 dimerinterface were also described in Schwartz, J. C. et al. (1 andreferences therein). In addition, some of the specific residuesparticipating in forming the human B7-1 dimer interface are underlinedin FIG. 12 .

The dimer interface of B7-1 is located within a region of theextracellular domain of human B7-1 and thus the present inventionprovides an isolated and purified peptide consisting of at least oneamino acid residue of the dimer interface within a region of theextracellular domain of human B7-1.

Therefore the presently disclosed subject matter relates to an isolatedand purified peptide comprising at least one amino acid residue of thedimer interface within a region of the extracellular domain of humanB7-1, said region consisting of the amino acid sequence denoted by SEQID NO: 14, namely, the amino acid sequence:

FCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLA EVTLSVKA.

The following amino acid residues participate in the formation of thedimer interface between monomeric B7-1 protein molecules: Val-15,Leu-29, Ala-30, Ser-48, Gly-49, Asp-50, Met-51, Lys-58, Asn-59, Arg-60,Thr-61, Ile-62, Phe-63, Asp-64, Ile-65, Thr-66, Val-72, Leu-74 andAla-75 based on the numbering of SEQ ID NO: 14.

The B7-1 related isolated and purified peptide according to the presentdisclosure thus consists of at least one of the amino acid residuesparticipate in the formation of the dimer interface between monomericB7-1 protein molecules recited above and at least 2 additional aminoacid residues at each of its C-terminus and/or N-terminus, wherein saidadditional amino acid residues are consecutive amino acid residues ofSEQ ID NO:14 immediately adjacent to said at least one amino acidresidue of said dimer interface in SEQ ID NO: 14, wherein said isolatedand purified peptide consists of from 3 to about 30 amino acid residues,and functional fragments and derivatives thereof.

In the above and other embodiments, the B7-1 related isolated andpurified peptide according to the present disclosure thus consists of atleast one of the amino acid residues participate in the formation of thedimer interface between monomeric B7-1 protein molecules recited aboveand from 2 to about 8, consecutive amino acid residues immediatelyadjacent thereto in SEQ ID NO:14, at each of its C-terminus and/orN-terminus, and functional derivatives thereof.

In other words, in some embodiments the isolated and purified peptideaccording to the invention comprises said at least one amino acidresidue of the crystallographic dimer interface within a region of theextracellular domain of human B7-1, said region consisting of the aminoacid sequence denoted by SEQ ID NO: 14, and from 2 to about 8, forexample 2 to 8, 3 to 8, 4 to 8, 5 to 8, 6 to 8 or 7 to 8, morespecifically 2, 3, 4, 5, 6, 7 or 8 additional amino acid residues at itsC-terminus and/or N-terminus, wherein said additional amino acidresidues are consecutive amino acid residues of SEQ ID NO:14 locatedimmediately adjacent to said at least one amino acid residue of saidcrystallographic dimer interface in SEQ ID NO:14, and functionalfragments and derivatives thereof.

In some embodiments, the B7-1 related isolated and purified peptideaccording to the invention consists of an isolated and purified peptideconsisting of an amino acid sequence selected from the amino acidsequences denoted by SEQ ID NO:28 (VKEVATLS), SEQ ID NO:30 (VEELAQTR),SEQ ID NO:32 (MSGDMNIW), SEQ ID NO:34 (SIVILALR), SEQ ID NO:36(YKNRTIFDIT), SEQ ID NO:38 (MNIWPEYK, denoted pB1-4), SEQ ID NO:40(KNRTIFDITN, denoted pB1-7) and SEQ ID NO:42 (DITNNLSIV, denoted pB1-6),SEQ ID NO:46 (SGDMNIWPEYKNRTIFDITNNLSIVILA) and SEQ ID NO:48 (YKNRTIFD,denoted pB1-8) and functional derivatives thereof.

In further embodiments, the B7-1 related isolated and purified peptideaccording to the invention consists of an isolated and purified peptideconsisting of an amino acid sequence selected from the amino acidsequences denoted by SEQ ID NO:38 (MNIWPEYK, denoted pB1-4), SEQ IDNO:40 (KNRTIFDITN, denoted pB1-7), SEQ ID NO:42 (DITNNLSIV, denotedpB1-6), SEQ ID NO:46 (SGDMNIWPEYKNRTIFDITNNLSIVILA) and SEQ ID NO:48(YKNRTIFD, denoted pB1-8) and functional derivatives thereof.

As indicated above, the presently disclosed subject matter providesisolated and purified peptides consisting of at least one amino acidresidue of the dimer interface within a region of the extracellulardomain of human B7-2, or an isolated and purified peptide consisting ofat least one amino acid residue of the dimer interface within a regionof the extracellular domain of the human B7-1, and functionalderivatives thereof. The terms “peptide”, “oligopeptide” or“polypeptide” as used herein refer to amino acid residues, connected bypeptide bonds.

More specifically, “amino acid sequence” or “peptide sequence” is theorder in which amino acid residues, connected by peptide bonds, lie inthe chain in peptides and proteins. The sequence is generally reportedfrom the N-terminal end containing free amino group to the C-terminalend containing amide.

The term “amino acids” as used herein refers to naturally occurring andsynthetic amino acids, as well as amino acid analogs and amino acidmimetics that can function in a manner similar to the naturallyoccurring amino acids. Naturally occurring amino acids are those encodedby the genetic code, as well as those amino acids that are latermodified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine.“Amino acid analogs” refers to compounds that have the same fundamentalchemical structure as a naturally occurring amino acid. Such analogshave modified R groups or modified peptide backbones, but retain thesame basic chemical structure as a naturally occurring amino acid.“Amino acid mimetics” refers to chemical compounds that have a structurethat is different from the general chemical structure of an amino acid,but that functions in a manner similar to a naturally occurring aminoacid. Amino acids may be referred to herein by either their commonlyknown three letter symbols or by the one-letter symbols recommended bythe IUPAC-IUB Biochemical Nomenclature Commission.

It should be noted that the polypeptides according to the invention canbe produced synthetically, or by recombinant DNA technology. Methods forproducing polypeptides peptides are well known in the art.

It should be noted that in addition to any of the peptides, furtherencompassed in the presently disclosed subject matter are any functionalfragments thereof. “functional fragments” within the scope of thesedisclosure are 3 to about 20 amino acid residues fragments of thedisclosed novel peptides, of which at least one amino acid residue is anamino acid residues of the dimer interface of human B7-2 (Thr-11,Leu-26, Ser-27, Leu-46, Gly-47, Lys-48, Glu-49, Phe-51, Met-59, Gly-60,Arg-61, Thr-62, Ser-63, Phe-64, Asp-65, Ser-66, Asp-67, Arg-72, His-74and Asn-75 of SEQ ID NO:13) for fragments of the present humanB7-2-derived peptides or at least one amino acid residue of the dimerinterface of human B7-1 (Val-15, Leu-29, Ala-30, Ser-48, Gly-49, Asp-50,Met-51, Lys-58, Asn-59, Arg-60, Thr-61, Ile-62, Phe-63, Asp-64, Ile-65,Thr-66, Val-72, Leu-74 and Ala-75 of SEQ ID NO:14), for fragments of thepresent human B7-1-derived peptides.

It should be noted that in addition to any of the peptides of theinvention, the invention further encompasses any functional derivativesthereof. The term “functional derivative” of “derivative” is used todefine amino acid sequences (peptides), with any insertions, deletions,substitutions and modifications to the amino acid sequences (peptides)that do not alter the activity of the original polypeptides. By the term“derivative” it is also referred to homologues, variants and analoguesthereof, as well as covalent modifications of a polypeptides madeaccording to the present invention.

The terms “fragments”, “derivatives” and “functional derivatives” asused herein mean any of the peptides of the invention, specifically hB-1mimetic peptides or hB-2 mimetic peptides as defined herein, with anyinsertions, deletions, substitutions and modifications to the peptidethat do not interfere with their ability to therapeutically affectbacterial and other infections, as well as inflammations associatedtherewith, as described herein.

In some embodiments, derivatives refer to peptides, which differ fromthe peptides specifically defined in the presently disclosed subjectmatter by insertions or deletions of amino acid residues. It should beappreciated that by the terms “insertions” or “deletions”, as usedherein it is meant any addition or deletion, respectively, of amino acidresidues to the polypeptides used by the invention, of between 1 to 50amino acid residues, between 1 to 1 amino acid residues, andspecifically, between 1 to 10 amino acid residues. More particularly,insertions or deletions may be of any one of 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 amino acids. It should be noted that the insertions or deletionsencompassed by the invention may occure in any position of the modifiedpeptide, as well as in the N- and/or C-terminus thereof.

By way of a non-limiting example, the term “modifications” as usedherein refers to derivatives of peptides according to the presentlydisclosed subject matter that are positively charged, negatively chargedor neutral. In addition, the peptides of the invention may be in theform of a dimer, a multimer or in a constrained conformation, which canbe attained by internal bridges, short-range cyclizations, extension orother chemical modifications.

Further, the peptides may be extended at the N- and/or C-terminusthereof with various identical or different amino acid residues. As anexample for such extension, the peptide may be extended at theN-terminus and/or C-terminus thereof with identical or differenthydrophobic amino acid residue/s which may be naturally occurring orsynthetic amino acid residue/s. A specific synthetic amino acid residuewith which the peptide may be extended at its N-terminus and/orC-terminus is D-alanine.

An additional example for such an extension may be provided by peptidesextended both at the N- and/or C-terminus thereof with a cysteineresidue. In some embodiments, the presently disclosed peptides can becoupled through their N-terminus to a lauryl-cysteine (LC) residueand/or through their C-terminus to a cysteine (C) residue. Naturally,such an extension may lead to a constrained conformation due to Cys-Cyscyclization resulting from the formation of a disulfide bond.

Another example may be the incorporation of an N-terminallysyl-palmitoyl tail, the lysine serving as linker and the palmitic acidas a hydrophobic anchor. In addition, the peptides may be extended byaromatic amino acid residue/s, which may be naturally occurring orsynthetic amino acid residue/s, for example a specific aromatic aminoacid residue may be tryptophan. The peptides may be extended at the N-and/or C-terminus thereof with various identical or different organicmoieties which are not naturally occurring or synthetic amino acids. Asan example for such extension, the peptide may be extended at the N-and/or C-terminus thereof with an N-acetyl group.

For every single peptide sequence used in the presently disclosedsubject matter and disclosed herein, also included is the correspondingretro-inverse sequence wherein the direction of the peptide chain hasbeen inverted and wherein all the amino acids belong to the D-series.

The presently disclosed subject matter also encompasses anysubstitutions of the peptides disclosed herein. Conservativesubstitution tables providing functionally similar amino acids are wellknown in the art. Such conservatively modified variants are in additionto and do not exclude polymorphic variants, interspecies homologues, andalleles and analogous peptides of the invention.

For example, substitutions may be made wherein an aliphatic amino acid(G, A, I, L, or V) is substituted with another member of the group, orsubstitution such as the substitution of one polar residue for another,such as arginine for lysine, glutamic for aspartic acid, or glutaminefor asparagine. Each of the following eight groups contains otherexemplary amino acids that are conservative substitutions for oneanother:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

More specifically, amino acid “substitutions” are the result ofreplacing one amino acid with another amino acid having similarstructural and/or chemical properties, i.e., conservative amino acidreplacements. Amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, nonpolar “hydrophobic” amino acids are selected from thegroup consisting of Valine (V), Isoleucine (I), Leucine (L), Methionine(M), Phenylalanine (F), Tryptophan (W), Cysteine (C), Alanine (A),Tyrosine (Y), Histidine (H), Threonine (T), Serine (S), Proline (P),Glycine (G), Arginine (R) and Lysine (K); “polar” amino acids areselected from the group consisting of Arginine (R), Lysine (K), Asparticacid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q); “positivelycharged” amino acids are selected form the group consisting of Arginine(R), Lysine (K) and Histidine (H) and wherein “acidic” amino acids areselected from the group consisting of Aspartic acid (D), Asparagine (N),Glutamic acid (E) and Glutamine (Q).

The presently disclosed subject matter also encompasses any homologues,variants and analogues of the peptides disclosed herein (either thepeptides derived from the dimer interface of the human B7-2 (alsoreferred to herein as “hB7-2 mimetic peptides”) or the peptides derivesfrom the dimer interface of the human B7-1 (also referred to herein as“hB7-1 mimetic peptides”) specifically defined by their amino acidsequence according to the presently disclosed subject matter.

In some embodiments, the functional derivative of the isolated andpurified peptide according to the presently disclosed subject matter isany one of:

-   -   i. said B7-2 mimetic peptide that is extended at the N terminus        and/or the C terminus thereof by 1 to 4 consecutive amino acid        residues present in immediately adjacent corresponding positions        of the amino acid sequence denoted by SEQ ID NO: 13;    -   ii. said B7-2 mimetic peptide that is extended at the N terminus        and/or the C terminus thereof:        -   (a) by cysteine or by lauryl cysteine;        -   (b) by an organic moiety that is not naturally occurring or            by a synthetic amino acid residue;        -   (c) by N-acetyl or lysyl-palmitoyl residue;        -   (d) by hydrophobic amino acid residue(s) which may be            naturally occurring or synthetic amino acid residues; or    -   iii. a dimer or multimer of any of the peptides of (i) and (ii);    -   iv. a constrained conformation of said B7-2 mimetic peptide;    -   v. any of said B7-2 mimetic peptides and their derivatives as        defined in (i) to (iv), that is modified by at least one        synthetic mutation selected from insertion, deletion,        substitution, provided that the modified peptide comprises at        least one amino acid residue of the said dimer interface;    -   wherein said derivative consists of from 3 to about 40 amino        acid residues.

In some other embodiments, the functional derivative of the isolated andpurified peptide according to the presently disclosed subject matter isany one of:

-   -   i. said B7-1 mimetic peptide that is extended at the N terminus        and/or the C terminus thereof by 1 to 4 consecutive amino acid        residues present in immediately adjacent corresponding positions        of the amino acid sequence denoted by SEQ ID NO: 14;    -   ii. said B7-1 mimetic peptide that is extended at the N terminus        and/or the C terminus thereof:        -   (a) by cysteine or by lauryl cysteine;        -   (b) by an organic moiety that is not naturally occurring or            by a synthetic amino acid residue;        -   (c) by N-acetyl or lysyl-palmitoyl residue;        -   (d) by hydrophobic amino acid residue(s) which may be            naturally occurring or synthetic amino acid residues; or    -   iii. a dimer or multimer of any of the peptides of (i) and (ii);    -   iv. a constrained conformation of said B7-1 mimetic peptide;    -   v. any of said B7-1 mimetic peptides and their derivatives as        defined in (i) to (iv), that is modified by at least one        synthetic mutation selected from insertion, deletion,        substitution, provided that the modified peptide comprises at        least one amino acid residue of the said dimer interface;    -   wherein said derivative consists of from 3 to about 40 amino        acid residues.

In some specific embodiments the isolated peptide according to presentlydisclosed subject matter is extended at its N terminus and/or at its Cterminus by a D-Ala amino acid residue.

In further embodiments the isolated peptide according to the presentlydisclosed subject matter is extended at its N terminus and/or at its Cterminus by the amino acid D-Ala and is selected from the groupconsisting of SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:19, SEQID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27 and SEQ ID NO:45 forpeptides derived from the dimer interface of hB7-2 and from the groupconsisting of SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35,SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:47 andSEQ ID NO: 49 for peptides derived from the dimer interface of hB7-1.

In some embodiments the isolated peptide as herein defined is any one ofa peptide consisting of the amino acid sequence (D-A)EKFDSVHSKYM(D-A) asdenoted by SEQ ID NO: 6 (also designated herein as peptide D-Ala-pB2-4),a peptide consisting of the amino acid sequence (D-A)DSDSWTLR(D-A) asdenoted by SEQ ID NO: 10 (also designated herein as peptide D-Ala-pB2-6)and a peptide consisting of the amino acid sequence (D-A)MGRTSFDSDS(D-A)as denoted by SEQ ID NO: 12 (also designated herein as peptideD-Ala-pB2-7).

In specific embodiments the isolated peptide as herein defined is of theamino acid sequence (D-A)EKFDSVHSKYM(D-A) as denoted by SEQ ID NO: 6(also designated herein as peptide D-Ala-pB2-4).

In other specific embodiments the isolated peptide as herein defined isof the amino acid sequence (D-A)DSDSWTLR(D-A) as denoted by SEQ ID NO:10 (also designated herein as peptide D-Ala-pB2-6).

In further specific embodiments the isolated peptide as herein definedis of the amino acid sequence (D-A)MGRTSFDSDS(D-A) as denoted by SEQ IDNO: 12 (also designated herein as peptide D-Ala-pB2-7).

The D-Ala residues were added to the hB7-2 mimetic peptides of thepresent disclosure at both their C- and N-termini, solely for conferringgreater protease resistance in biological assays. The terms “D-Ala-” and“(D-A)” are used herein interchangeably. In further specific embodimentsthe isolated peptide as herein defined is any one of a peptideconsisting of the amino acid sequence (D-A)MNIWPEYK(D-A) as denoted bySEQ ID NO: 39 (also designated D-Ala-pB1-4), a peptide consisting of theamino acid sequence (D-A)KNRTIFDITN(D-A) as denoted by SEQ ID NO:41(also designated D-Ala-pB1-7), a peptide consisting of the amino acidsequence (D-Ala)DITNNLSIV(D-Ala) as denoted by SEQ ID NO:43 (alsodesignated D-Ala-pB1-6), and a peptide consisting of the amino acidsequence (D-Ala)YKNRTIFD(D-Ala) denoted by SEQ ID NO:49 (also designatedD-Ala-pB1-8).

The D-Ala residues are added to the hB7-1 mimetic peptides of thepresent disclosure at both their C- and N-termini, solely for conferringgreater protease resistance in biological assays.

The isolated and purified peptides according to all aspects andembodiments of the present disclosure, and their functional fragmentsand derivatives, are synthetic, non-naturally occurring, man-madepeptides. The peptides are synthetic, non-naturally occurring, man-madepeptides also when any or all of their amino acids are naturallyoccurring per se.

The terms “synthetic peptide/s”, “non-naturally occurring peptide/s” and“man-made peptide/s” as used herein are to be taken to mean peptidesthat cannot be found in nature as such at their specific length andamino acid sequence.

As used herein, the term “isolated”, in the context of the presentlydisclosed subject matter, means amino acid sequences which may havepartial or complete homology to naturally occurring peptides,polypeptides are proteins, but are of limited lengths, and differ, atleast in length, from such naturally occurring sequences. For example,isolated peptides according to the presently disclosed subject mattercan be specific, defined fragments of a region of the extracellulardomain of human B7-2 or human B7-1, of shorter length and are notexpected to be found as such at their natural milieu. Isolated peptidesare generally prepared synthetically, by chemical synthesis, chemicallyor enzymatically, by cleaving a naturally occurring sequence, orsometimes by recombinant techniques.

As such, “isolated” does not necessarily reflect the extent to which theamino acid molecules have been purified. However, it will be understoodthat such molecules that have been purified to some degree are“isolated”. If said molecules do not exist in a natural milieu, i.e.they do not exist in nature, the molecule is “isolated” regardless ofwhere it is present.

Furthermore, the term “purified” or “substantially purified”, whenapplied to peptides, denotes that the peptides are essentially free ofother cellular components with which they are associated in the naturalstate. Rather, the peptides are in a homogeneous state, although theycan be either dry or in solution. Purity and homogeneity are typicallydetermined using analytical chemistry techniques such as polyacrylamidegel electrophoresis or high performance liquid chromatography. A peptidemolecule which is the predominant species present in a preparation ispurified or substantially purified.

The present disclosure further provides a pharmaceutical compositioncomprising as an active ingredient at least one isolated and purifiedpeptide as defined herein optionally further comprising apharmaceutically acceptable carrier, diluent, adjuvant and/or excipient.

In some embodiments the pharmaceutical composition as herein definedcomprises as an active ingredient at least one, or at least two, or atleast three or more isolated and purified peptides as herein defined,optionally further comprising a pharmaceutically acceptable carrier,diluent, adjuvant and/or excipient.

As shown below in Example 3 and FIGS. 5A and 5B, mice challenged withSEB, that were administered with peptides D-Ala-pB2-4, D-Ala-pB2-6 andD-Ala-pB2-7 30 minutes before SEB challenge, survived SEB challenge(FIG. 5A). Notably, the peptide D-Ala-pB2-7 protected when present inonly 2- to 3-fold molar excess over SEB (FIG. 5B). This high efficacysupports a critical role for the B7-2 dimer interface in mediating thedeleterious response to superantigens.

Therefore in another one of its aspects the present disclosure providesthe disclosed peptides and pharmaceutical compositions comprising themas herein defined, for eliciting protective immunity against at leastone of sepsis, toxic shock, septic shock, severe sepsis, incapacitationand resulting death, that are induced by a bacterial pathogen, a mixtureof bacterial pathogens and/or a toxic bacterial component.

The term “eliciting protective immunity” as herein defined meansprotecting animals infected with lethal pathogenic bacteria and/orlethal toxic bacterial components against the toxic shock induced,including septic shock, sepsis and/or severe sepsis, incapacitation andresulting death from any thereof, thereby leading to rescue and survivalof infected/challenged animal from the induced toxic shock.

Without being bound by theory, it is also suggested that a result oftreatment and rescue of animals infected with lethal pathogenic bacteriaand/or lethal toxic bacterial components may be the conferring oflong-term protective immunity against toxic shock, septic shock, severesepsis, incapacitation and resulting death, that are induced by lethalbacterial pathogen/s and/or toxic bacterial component/s and theinfected/challenged animals not only survive immediate shock, butacquire protective immunity against any further toxin challenges. Thisis because by blocking the ability of the toxin to induce a cellularimmune response leading to toxic shock, the antagonist peptides of thepresent disclosure may allow the superantigen to induce an immuneresponse directed against itself.

By another one of its aspects the present disclosure provides a peptideas herein defined for use in a method for eliciting in a human subjectin need protective immunity against at least one of sepsis, toxic shock,septic shock, severe sepsis, incapacitation and resulting death, thatare induced by a bacterial pathogen, a mixture of bacterial pathogensand/or a toxic bacterial component.

By still another one of its aspects the present disclosure provides apharmaceutical composition as herein defined for use in a method foreliciting in a human subject in need protective immunity against atleast one of sepsis, toxic shock, septic shock, severe sepsis,incapacitation and resulting death, that are induced by a bacterialpathogen, a mixture of bacterial pathogens and/or a toxic bacterialcomponent.

The pharmaceutical compositions of the presently disclosed subjectmatter generally comprise a buffering agent, an agent which adjusts theosmolarity thereof, and optionally, one or more pharmaceuticallyacceptable carriers, diluent, adjuvant and/or excipients and/oradditives as known in the art. Supplementary active ingredients can alsobe incorporated into the compositions. The carrier can be solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), suitable mixtures thereof, and vegetable oils. The properfluidity can be maintained, for example, by the use of a coating, suchas lecithin, by the maintenance of the required particle size in thecase of dispersion and by the use of surfactants. The carrier, diluent,adjuvant and/or excipient do not interfere with the activity of thepeptide.

Salts and esters of the presently disclosed peptides are alsoencompassed by the presently disclosed subject matter. The term “salts”as herein defined refers to a pharmaceutically acceptable salt, e.g.,non-toxic alkali metal, alkaline earth metal, and ammonium saltscommonly used in the pharmaceutical industry including the sodium,potassium, lithium, calcium, magnesium, barium, ammonium, and protaminezinc salts, which are prepared by methods well known in the art. Theterm also includes non-toxic acid addition salts, which are generallyprepared by reacting the active compounds used herein with a suitableorganic or inorganic acid. Specific acid addition salts may behydrochloride, acetate, maleate, malate, tartrate, salicylate, citrateor malonate salts, and solvates, e.g. hydrates thereof.

The term “ester” as herein defined refers to a pharmaceuticallyacceptable ester, e.g. esters which retain, upon hydrolysis of the esterbond, the biological effectiveness and properties of the carboxylic acidor alcohol and are not biologically or otherwise undesirable. Generally,ester formation can be accomplished via conventional synthetictechniques.

In specific embodiments, said pharmaceutical composition can be insustained- or controlled-release form, or in a combinedsustained/controlled-release and immediate release forms.

In the above and other embodiments of the disclosed subject matter, thepeptide may be comprised in a pharmaceutical unit dosage form, saiddosage form optionally further comprising at least one ofphysiologically compatible additives, carriers, peptide stabilizers,diluents and excipients. For example, said dosage form may optionallyfurther comprise protease inhibitors.

In the above and other aspects and embodiments of the disclosed subjectmatter, the pharmaceutical composition can be used for the treatment ofat least one of a bacterial infection and acute inflammation associatedtherewith in a human subject.

Bacterial infections in human are generally induced by at least one ofGram-positive bacteria, Gram-negative bacteria and toxic bacterialcomponents. Where infection is with more than one bacterium, it may bereferred to as a polymicrobial infection, as defined below.

Thus in the above and other embodiments of the disclosed subject matter,the pharmaceutical composition can be used for the treatment of at leastone of bacterial infection and acute inflammation associated therewith,induced by at least one of Gram-positive bacteria, Gram-negativebacteria, and at least one toxic bacterial component.

In specific embodiments the peptide or the pharmaceutical compositioncomprising as an active ingredient at least one isolated and purifiedpeptide as herein defined is for use in a method of treating at leastone of bacterial infection and acute inflammation associated therewithin a human subject in need, wherein said pharmaceutical compositionoptionally further comprises a pharmaceutically acceptable carrier,diluent, adjuvant and/or excipient.

Gram-negative bacteria as herein defined and as known in the art arebacteria that do not retain crystal violet dye in the Gram stainingprotocol, due to the existence of an outer membrane preventing thepenetration of the stain. In contrast, Gram positive bacteria willretain the crystal violet dye when washed in a decolorizing solution.The pathogenic capability of Gram-negative bacteria is often associatedwith certain components of Gram-negative cell envelope, in particular,the lipopolysaccharides (LPS) layer. In humans, LPS triggers an innateimmune response characterized by cytokine production and immune systemactivation, which is often associated with inflammation.

Thus in the above and other embodiments of the disclosed subject matter,the Gram-negative bacteria can be, but are not limited to, any one ofproteobacteria, Escherichia coli, Salmonella, Shigella,Enterobacteriaceae, Pseudomonas, Moraxella, Helicobacter, Bdellovibrio,Stenotrophomonas, acetic acid bacteria, Legionella,alpha-proteobacteria, Wolbachia, Gram-negative cocci, Neisseria species,Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis,Gram-negative bacilli, Hemophilus influenzae, Klebsiella pneumoniae,Legionella pneumophila, Pseudomonas aeruginosa, Proteus mirabilis,Enterobacter cloacae, Serratia marcescens, Helicobacter pylori,Salmonella enteritidis, Salmonella typhi, Acinetobacter baumannii,Francisella tularemia, Vibrio, vulnificus, cholerae, fluvialis,parahemolyticus, alginolyticus, Photobacter damsela, Aeromonashydrophila, Clostridium perfringens, Clostridium histolyticum,Porphyromonas/prevotella sp. Prevotella Intermedia, Prevotella Buccae,Prevotella sp., Bacteroides uniformis and NDM-1 bacterial strains.

Gram-positive bacteria as known in the art are herein defined asbacteria that are stained dark blue or violet by Gram staining, incontrast to Gram-negative bacteria, as recited above. As a non-limitingexample, Group A streptococci (GAS) are Gram positive bacteriaresponsible for a wide range of both invasive and non-invasiveinfections. GAS produce a range of superantigenic toxins which arebelieved to be important in the pathogenesis of invasive streptococcalinfections such as streptococcal toxic shock syndrome.

Thus in the above and other embodiments of the disclosed subject matter,the Gram-positive bacteria are selected from the group consisting ofGroup A streptococcus, S. pyogenes, S. pneumonia, Group B strep,Enterococcus faecalis, Group D streptococcus, Group G streptococcus,Streptococcus viridans, Streptococcus milleri, Propionibacterium sp.,Enterococcus faecium, Peptostreptococcus sp., StreptococcusMicroaerophilic, Lactobacillus sp., Staphylococcus Epidermis andStaphylococcus aureus.

The bacterium Staphylococcus aureus (also referred to as S. aureus orStaph. aureus) is a facultative anaerobic Gram-positive coccalbacterium, which is frequently found in the human respiratory tract andon the skin. Although S. aureus is not always pathogenic, it is a commoncause of skin infections (e.g. boils), respiratory disease (e.g.sinusitis), and food poisoning. Disease-associated strains often promoteinfections by producing potent protein toxins. The emergence ofantibiotic-resistant forms of pathogenic S. aureus is a worldwideproblem in clinical medicine.

Streptococcus pyogenes is a spherical, Gram-positive bacterium that isthe cause of group A streptococcal infections. S. pyogenes displaysstreptococcal group A antigen on its cell wall. It is estimated thatthere are more than 700 million infections world-wide each year and over650,000 cases of severe, invasive infections that have a mortality rateof 25%. Early recognition and treatment are critical and failure indiagnosis may result in sepsis and death.

The term “polymicrobial infection” as used herein is to be taken to meanan infection consisting of/induced by several species of bacteria. Thebacterial infection may be caused by a mixture of Gram-positivebacteria, by a mixture of Gram-negative bacteria or by a mixture of bothGram-positive and Gram-negative bacteria. A polymicrobial infection canalso be caused by a mixture of aerobic bacteria, anaerobic bacteria orboth.

Therefore, in the above and other embodiments of the disclosed subjectmatter said polymicrobial infection is induced by Gram-positivebacteria, Gram-negative bacteria, or a combination thereof.

Bacterial toxins and other toxic bacterial components are well known inthe art. Bacteria generate toxins which can be classified as eitherexotoxins or endotoxins. While exotoxins are generated and activelysecreted, endotoxins remain part of the bacteria. The response of thehost to an endotoxin can involve severe inflammation. While generallythe inflammation process is usually clinically considered beneficial tothe infected host, when the reaction is severe, it may lead to sepsis.In particular, the terms “superantigen toxin” or “superantigen” (alsoreferred to as “SAgs”) as used herein refers to a class of bacterialpyrogenic exotoxins that cause non-specific activation of T-cellsresulting in polyclonal T cell activation and massive cytokine release.

Staphylococcal enterotoxin B (SEB) is an enterotoxin produced by thebacterium Staphylococcus aureus, which is a Gram-positive coccalbacterium. It is a common cause of food poisoning, with severe diarrhea,nausea and intestinal cramping often starting within a few hours ofingestion. Being quite stable, the toxin may remain active even afterthe contaminating bacteria are killed. It can withstand boiling at 100°C. for a few minutes. SEB is regarded as a superantigen, causing theimmune system to release a large amount of cytokines that lead tosignificant inflammation.

Toxic shock syndrome toxin (TSST) is a superantigen with a size of 22KDa produced by 5 to 25% of Staphylococcus aureus isolates. It causestoxic shock syndrome (TSS) by stimulating the release of large amountsof interleukin-1, interleukin-2, and tumor necrosis factor (TNF). Ingeneral, the toxin is not produced by bacteria growing in the blood;rather, it is produced at the local site of an infection, and thenenters the blood stream.

TSST-1 is a prototype superantigen secreted by a Staphylococcus aureusbacterium strain in susceptible hosts, acts on the vascular system bycausing inflammation, fever, and shock. The bacterium strain thatproduces TSST-1 lives mostly in the vagina of infected women (one-thirdof all TSS cases have been found in men).

Streptococcal mitogenic exotoxin Z (SMEZ) is one of the most potentsuperantigenic toxins produced by the streptococcal genome. The geneencoding SMEZ is present in all GAS strains studied. Although noclinical data are yet available demonstrating an association of SMEZwith GAS disease, the prevalence, potency, and antigenic variation shownby SMEZ suggest that this superantigen may have an important function inthe pathogenesis of streptococcal disease.

As used herein, the term “lipopolysaccharide” (LPS), or “lipoglycan”refers to a large molecule consisting of a lipid and a polysaccharidejoined by a covalent bond; LPS are found in the outer membrane ofGram-negative bacteria, act as endotoxins and elicit strong immuneresponses in animals. LPS is the major component of the Bacterial cellwall of Gram-negative bacteria, contributing greatly to the structuralintegrity thereof. LPS is an endotoxin, and induces a strong responsefrom normal animal immune systems.

Therefore, in all aspects and embodiments of the disclosed subjectmatter said toxic bacterial components are selected from the groupconsisting of exotoxins, endotoxins, superantigen toxins, pathogenassociated molecular patterns (PAMPs), Damage Associated MolecularPattern molecules (DAMPs), lipopolysaccharides or toxic componentsthereof, molecules that are associated with groups of pathogens that arerecognized by cells of the innate immune system and molecules that areassociated with groups of pathogens that are recognized by Toll-likereceptors (TLRs), but are not limited thereto.

Thus the pharmaceutical composition according to the presently disclosedsubject matter can be used in the treatment of infections induced byGram-negative bacteria selected from the group consisting ofproteobacteria, Escherichia coli, Salmonella, Shigella,Enterobacteriaceae, Pseudomonas, Moraxella, Helicobacter, Bdellovibrio,Stenotrophomonas, acetic acid bacteria, Legionella,alpha-proteobacteria, Wolbachia, Gram-negative cocci, Neisseria species,Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis,Gram-negative bacilli, Hemophilus influenzae, Klebsiella pneumoniae,Legionella pneumophila, Pseudomonas aeruginosa, Proteus mirabilis,Enterobacter cloacae, Serratia marcescens, Helicobacter pylori,Salmonella enteritidis, Salmonella typhi, Acinetobacter baumannii,Francisella tularemia, Vibrio, vulnificus, cholerae, fluvialis,parahemolyticus, alginolyticus, Photobacter damsela, Aeromonashydrophila, Clostridium perfringens, Clostridium histolyticum,Porphyromonas/prevotella sp. Prevotella Intermedia, Prevotella Buccae,Prevotella sp., Bacteroides uniformis and NDM-1 bacterial strains,Gram-positive bacteria selected from the group consisting of Group Astreptococcus, S. pyogenes, S. pneumonia, Group B strep, Enterococcusfaecalis, Group D streptococcus, Group G streptococcus, Streptococcusviridans, Streptococcus milleri, Propionibacterium sp., Enterococcusfaecium, Peptostreptococcus sp., Streptococcus Microaerophilic,Lactobacillus sp., Staphylococcus Epidermis and Staphylococcus aureus,or a combination thereof, toxic bacterial components selected from thegroup consisting of exotoxins, endotoxins, superantigenic toxins,pathogen associated molecular patterns (PAMPs), Damage AssociatedMolecular Pattern molecules (DAMPs), lipopolysaccharides, peptidoglycansor toxic components thereof, molecules that are associated with groupsof pathogens that are recognized by cells of the innate immune systemand molecules that are associated with groups of pathogens that arerecognized by Toll-like receptors (TLRs).

Is specific embodiments, the at least one toxic bacterial component is asuperantigenic toxin. In further specific embodiments the pharmaceuticalcomposition as herein defined is wherein the at least one toxicbacterial component is a superantigenic toxin.

In the above and other embodiments of the disclosed subject matter,administration may be performed by any of the following routes: oraladministration, intravenous, intramuscular, intraperitoneal, intranasal,intrathecal subcutaneous injection or said administration is byinhalation. Intravenous administration may be continuous administration,specifically over a period of from about 10 to about 30 minutes.Intravenous administration may alternatively be push administration.

In the above and other embodiments of the disclosed subject matter thepharmaceutical composition according to the invention is for any one oforal administration and parenteral administration.

In the above and other embodiments of the disclosed subject matter thepharmaceutical composition according to the invention is for any one oforal administration and intravenous, intramuscular, intraperitoneal,intranasal, intrathecal, subcutaneous injection or said administrationis by inhalation.

In further embodiments of the presently disclosed subject matter theparenteral administration is any one of intravenous, intramuscular,intraperitoneal, intranasal, intrathecal, subcutaneous injection or saidadministration is by inhalation.

Further disclosed is a method of treating at least one of bacterialinfection and acute inflammation associated therewith in a human subjectin need, comprising administering to said subject a therapeuticallyeffective amount of at least one peptide as defined herein or acomposition comprising the same as defined herein. This method is alsoreferred to herein as “method of treating”.

The term “treat” or “treatment” or forms thereof as herein defined meansto prevent worsening or arrest or alleviate or cure the disease orcondition in a subject in need thereof.

The term “therapeutically effective amount” (or amounts) of the peptidefor purposes herein defined is determined by such considerations as areknown in the art in order to cure or at least arrest or at leastalleviate the medical condition.

In some embodiments the therapeutically effective amount as hereindefined is from about 0.1 to about 60 μg/Kg body weight of said subject.

Further disclosed is a method for eliciting in a human subject in needprotective immunity against at least one of sepsis, toxic shock, septicshock, severe sepsis, incapacitation and resulting death, that areinduced by a bacterial pathogen, a mixture of bacterial pathogens and/orat least one toxic bacterial component, said method comprisingadministering to said subject an immunologically effective amount of apeptide as defined herein or of a pharmaceutically acceptablecomposition comprising the same. In this method, the at least one toxicbacterial component may be selected from the group consisting ofexotoxins, endotoxins, superantigenic toxins, pathogen associatedmolecular patterns (PAMPs), Damage Associated Molecular Patternmolecules (DAMPs), lipopolysaccharides, peptidoglycans or toxiccomponents thereof, molecules that are associated with groups ofpathogens that are recognized by cells of the innate immune system andmolecules that are associated with groups of pathogens that arerecognized by Toll-like receptors (TLRs), specifically superantigenictoxins. In specific embodiments the toxic bacterial component is, butnot limited to, SEB, SMEZ or TSST-1. Efficacy of presently disclosedpeptides against these toxins is shown in Example 1. This method is alsoreferred to herein as “method of conferring immunity”.

The term “immunologically effective amount” (or amounts) of the peptidemean any amount sufficient to confer immunity against sepsis, toxicshock, septic shock, severe sepsis, incapacitation and resulting deathinduced by pathogenic bacteria and/or toxic bacterial component/s and isdetermined by such considerations as are known in the art.

As shown in Example 3 below, the peptides according to the invention,e.g. peptides D-Ala-pB2-4, D-Ala-pB2-6 and D-Ala-pB2-7, were shown toblock superantigen lethality in mice when the peptides were administeredat a therapeutically effective amount ranging from 0.2 to 1 μg per mouse(see FIGS. 5A and 5B). This effective amount in a mouse weighing 25 grcorresponds to about 40 μg/kg, the human equivalent dose (HED) of whichbeing 0.65 to 3.25 μg/kg.

Specific ranges used by the method of treating and method of conferringimmunity disclosed herein can be from about 0.1 μg to about 60 μgpeptide/kg body weight of said subject. Thus, in the above and otherembodiments of the disclosed subject matter, the peptide according tothe present disclosure is administered to said human subject in anamount of from about 0.1 to about 60 μg peptide/kg body weight of saidsubject.

Thus, the amount can be from 0.1 μg to 60 μg peptide/kg body weight ofsaid subject, such as 0.1-60.0, 0.1-55.0, 0.1-40.0, 0.1-35.0, 0.1-30.0,0.1-25.0, 0.1-20.0, 0.1-15.0, 0.1-10.0, 0.1-9.0, 0.1-8.0, 0.1-7.0,0.1-6.0, 0.1-5.0, 0.1-4.0, 0.1-3.0, 0.1-2.0, 0.1-1.0, 0.1-0.75, 0.1-0.5,0.1-0.25. Specifically, the therapeutically effective amount may be anyone of 0.25, 0.5, 0.65, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5,2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, 5.0, 5.25, 5.5, 5.75,6.0, 6.25, 6.5, 6.75, 7.0, 7.25, 7.5, 7.75, 8.0, 8.25, 8.5, 8.75, 9.0,9.25, 9.5, 9.75, 10.0, 12.5, 15.0, 17.5, 20.0, 25.0, 30.0, 35.0, 40.0,45.0, 50.0, 55.0 or 60.0 μg peptide/kg body weight of said subject.

It is to be noted that the amount of the peptide to be administered mayvary by about 5-25%, in consideration of the molecular weight and otherfeatures of a specific peptide. Thus the term “about” as herein definedrefers to a fluctuation of 5-25% of the amount as herein defined.

In the above and other embodiments of the disclosed subject matter, thepeptide for use in treatment of bacterial infection and/or acuteinflammation associated therewith according to the present disclosuremay be administered at a suitable time post onset of said at least oneof infection and acute inflammation associated therewith. Alternativelyor additionally, the peptide for use according to the present disclosuremay be administered immediately following the onset of said infection oracute inflammation associated therewith. The peptide according to thepresent disclosure may be administered during the acute phase of theinfection, and if necessary, also thereafter, as may be determined bythe attending physician.

The term “onset” refers to any time point between the time of infectionof said human subject or the time of beginning of its clinicalmanifestation or the manifestation of acute inflammation associated withor resulting from said infection and the time of diagnosis of any of theinfection and inflammation by a skilled member of attending medicalstaff, and any time there-between or thereafter, in which treatment inaccordance with the present disclosure is professionally assigned tosaid subject.

As used herein, the term “human subject in need” is to be taken to meana human suffering from at least one of infection and acute inflammationassociated therewith as herein defined. The “human subject in need” maybe a human subject suffering from sepsis, toxic shock, septic shock,severe sepsis and/or incapacitation that may result in death, that areinduced by lethal bacterial pathogen/s and/or toxic bacterialcomponent/s of bacterial pathogen/s. The “human subject in need” mayalso be a human at risk of being inflicted by bacterial infection andacute inflammation associated therewith.

In the above and other embodiments of the disclosed subject matter, thepharmaceutical composition or method of treating according to theinvention optionally further comprises administering to said humansubject in need an additional therapeutic agent.

In the above and other embodiments of the disclosed subject matter theadditional therapeutic agent can be any one of antibacterial agent,antiviral agent, antifungal agent, antibiotic agent, bacteriostatic andbactericidal agent, steroid and antimicrobial agent.

In the above and other embodiments of the disclosed subject matter, saidpeptide and said additional other therapeutically effective agent areadministered simultaneously. Alternatively or additionally, said peptideand said additional other therapeutically effective agent areadministered at different time points, at different intervals betweenadministrations, for different durations of time, or in a differentorder.

For example, treatment may commence with administration of both thepeptide and the additional other agent, and administration of theadditional agent may be ceased before or after the administration of thepeptide.

In the above method of conferring to a subject protective immunityagainst at least one of sepsis, toxic shock, septic shock, severesepsis, incapacitation and resulting death, that are induced by abacterial pathogen, a mixture of bacterial pathogens and/or at least onetoxic bacterial component, the peptide according to the presentdisclosure may specifically be administered at a suitable time beforechallenge with said bacterial pathogen/s and/or toxic bacterialcomponent/s, or thereafter. When administered before challenge, thepeptide may be administered at about 30, 25, 20, 15 minutes or lessbefore challenge. When administered after challenge, the peptide isadministered immediately or shortly thereafter.

It is appreciated that certain features of the presently disclosedsubject matter, which are, for clarity, described in the context ofseparate embodiments, may also be provided in combination in a singleembodiment. Conversely, various features of the invention, which are,for brevity, described in the context of a single embodiment, may alsobe provided separately or in any suitable sub combination or as suitablein any other described embodiment of the invention. Certain featuresdescribed in the context of various embodiments are not to be consideredessential features of those embodiments, unless the embodiment isinoperative without those elements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

Disclosed and described, it is to be understood that this invention isnot limited to the particular examples, methods steps, and compositionsdisclosed herein as such methods steps and compositions may varysomewhat. It is also to be understood that the terminology used hereinis used for the purpose of describing particular embodiments only andnot intended to be limiting since the scope of the present inventionwill be limited only by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise.

EXAMPLES

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe claimed invention in any way.

Standard molecular biology protocols known in the art not specificallydescribed herein are generally followed essentially as in Sambrook &Russell, 2001.

Standard medicinal chemistry methods known in the art not specificallydescribed herein are generally followed essentially in the series“Comprehensive Medicinal Chemistry” by various authors and editors,published by Pergamon Press.

Experimental Procedures

Peptides

Peptides were synthesized using fluoronyl-methoxycarbonyl chemistry,cleaved and the side chain deprotected with triflouroacetic acid.Peptides were >95% pure by high-pressure liquid chromatography;molecular weight was verified by MALDI-TOF mass spectrometry. Peptideswere abutted with D-Ala (D-Alanine) at both termini for greater proteaseresistance in biological assays and with Cys (Cysteine) for SurfacePlasmon Resonance (SPR). B7-2 peptides were dissolved readily into RPMI1640 tissue culture medium.

Antibodies

αSEB monoclonal antibody (Toxin Technology: clone MB2B33), horseradishperoxidase-conjugated goat anti-mouse IgG or donkey anti-goat (KPL),mouse monoclonal αCD28 (clone 37407), αCD3 (clone UCHT1), goatpolyclonal anti-CD28 and anti-B7-2 (R&D Systems) antibodies were used.Binding of SEB to immobilized B7-2-Fc and ribonuclease A was assayed inenzyme-linked immunosorbent assays using corresponding horseradishperoxidase-conjugated mouse anti-SEB monoclonal antibody.

Induction of Cytokine Gene Expression

Human peripheral blood mononuclear cells (PBMC) from healthy humandonors were separated on Ficoll Paque (Amersham), washed twice with 50ml of RPMI 1640 medium, resuspended at 4×10⁶ cells/ml and cultured inthis medium supplemented with 2% fetal calf serum, 2 mM glutamine, 10 mMMEM nonspecific amino acids, 100 mM Na-pyruvate, 10 mM Hepes pH 7.2,5×10⁻⁵ M 2-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycinand 5 μg/ml nystatin. Staphylococcal enterotoxin B (SEB, Lot 1430,Department of Toxinology, U.S. Army Medical Research Institute ofInfectious Diseases) (10, 12) was added to a final concentration of 100ng/ml. Mouse antihuman monoclonal antibodies αCD3 (clone UCHT1; 100ng/ml) and αCD28 (clone 37407; 2.5 g/ml) (R&D Systems, Minneapolis,Minn.) were used as inducers. The E. coli Lipopolysaccharide (LPS)0111:B4 for studies with human PBMC was obtained from Sigma Aldrich (St.Louis, Mo.). Secreted cytokines were quantitated in triplicate withQuantikine ELISA kits (R&D Systems) and are presented as means±SEM.

B7-2 Expression Vectors

A vector expressing B7-2 was generated by cDNA synthesis of human CD86(NM_175862) from total human PBMC RNA using Verso RT-PCR kit (ABgene).CD86 cDNA was generated using KOD polymerase (Novagen) with thephosphorylated PCR primers 5′-GACGTCGACGGAAGGCTTGCACAGGGT (denoted bySEQ ID NO:51) and 5′-CACGCGGCCGCCCAGGTCATGAGCCATTAAGC (denoted by SEQ IDNO:52). The PCR product was inserted into pEGFP-N3 DNA (Clontech) thathad been digested with SalII and NotI and lacked the GFP region, usingFast-Link DNA Ligation Kit (Epicentre).

Vectors expressing B7-2 fused C-terminally to GFP or Cherry weregenerated from B7-2 cDNA vector template with the phosphorylated PCRprimers 5′-TACTCGAGATGGGACTGAGTAACATTC (denoted by SEQ ID NO:53) and5′-GTCCGCGGTGAAGCATGTACACTTTTGTCG (denoted by SEQ ID NO:54), deletingthe B7-2 termination codon. Upon digestion with Xhol and SacII, the PCRproduct was inserted either into pEGFP-N3 DNA or pmCherry-N1 DNA(Clontech). B7-2C/Cherry vector was generated from B7-2/Cherry templateusing the primers 5′-GTCTCTCGTCCTTCCGG (denoted by SEQ ID NO:55) and5′-CTAACTTCAGTCAACCTG (denoted by SEQ ID NO:56).

TABLE 1 Sequences of primers SEQ ID NO: Sequence Description SEQ IDGACGTCGACGGAAGGCT Primer for B7-2 cloning NO: 51 TGCACAGGGT SEQ IDCACGCGGCCGCCCAGGT Primer for B7-2 cloning NO: 52 CATGAGCCATTAAGC SEQ IDTACTCGAGATGGGACTG Primer for preparing B7- NO: 53 AGTAACATTC2-GFP fusion construct SEQ ID GTCCGCGGTGAAGCATG Primer for preparing B7-NO: 54 TACACTTTTGTCG 2-GFP fusion construct SEQ ID GTCTCTCGTCCTTCCGGPrimer for preparing B7- NO: 55 2C/Cherry vector SEQ IDCTAACTTCAGTCAACCT Primer for preparing B7- NO: 56 G 2C/Cherry vectorCD28B7-2 Interaction

In order to assay the effect of SEB on binding of B7-2 to CD28 on thecell, HEK293T cells were transfected to express cell-surface CD28 (10)or with empty vector expressing GFP. After 36 hours, the cells wereincubated for 45 minutes with 0.2 μg/ml soluble B7-2 in the absence orpresence of SEB. After three washes with cold phosphate-buffered saline,cells were lysed. Equal amounts of total cell protein (Bradford assay)were subjected to 10% polyacrylamide gel electrophoresis (PAGE) andwestern blotting to show binding of B7-2 and expression of CD28 by thecells. Conversely, the effect of SEB on binding of CD28 to B7-2 on thecell was assayed by transfecting HEK293T cells to express cell-surfaceB7-2. After 36 hours, the cells were incubated for 45 minutes with 0.2μg/ml soluble CD28 in the absence or presence of SEB. After three washesas above, cells were lysed. Equal amounts of total cell protein weresubjected to 10% PAGE and Western blotting to show binding of CD28 andexpression of B7-2 by the cells.

Recombinant Superantigens

Chromosomal DNA isolated from S. aureus COL, from a TSST-1-producingstrain of S. aureus, and from a SMEZ-producing strain of Streptococcuspyogenes was used to clone wt SEB, TSST-1 and SMEZ genes, respectively,into pHTT7K (14). Each one of the above genes was expressed in E. colias the mature proteins with an N-terminal His₆-tag. Inserts wereverified by DNA sequencing. Total protein was loaded onto a His-Bindcolumn (Novagen) and eluted stepwise with imidazole. Recombinantproteins recovered after dialysis were >98% pure on SDS-PAGE and >98%homogeneous as monomer upon analytical gel filtration through a 1×30 cmSuperdex 75 column calibrated with molecular weight standards (GEHealthcare-Amersham Pharmacia) from which protein was eluted at a flowrate of 1 ml/min. Recombinant SEB was lethal to mice at 10 μg/ml.

Surface Plasmon Resonance Spectroscopy

Proteins and peptides were diluted to 10-200 μg/ml in 10 mM Na acetatepH 4.0 and immobilized on a CM5 sensorchip using amine coupling kit andamine-thiol coupling kit (BIAcore), respectively. Analytes were injectedat 20 l/min in 25 mM HEPES pH 7.4, 150 mM NaCl, 3.4 mM EDTA, and 0.005%surfactant P20 under low ligand density conditions that minimize masstransfer limitations; a maximal binding capacity of the immobilizedligand in the range of 50-150 response units enables measurement ofbinding kinetics. Regeneration was with 50 mM phosphoric acid. Kineticanalyses were performed at 25° C. in a BIAcore 3000 instrument,deducting the control flow cell signal from the binding signal. Analytecurves were run in duplicate; representative results are shown.BIAevaluation 3.1 software was used to determine K_(D) in the linearligand concentration range (1:1 Langmuir binding). Criteria for K_(D)determination for complexes were standard error as percent of k_(a) andk_(d); λ² values well below 2 show the quality of fit between calculatedand observed binding values and attest to the purity of the ligandsexamined. Fitting residuals were within the range of ±2, validating thegoodness of fit. Human IgG (Jackson Laboratories) and ribonuclease A(Sigma) served as controls.

Mouse Lethality Assay

Female BALB/c mice (10-12 weeks; Harlan) were challenged byintraperitoneal injection of SEB and 20 mg D-galactosamine to sensitizethe animals to superantigens (12). Antagonist peptides were injectedintraperitoneally 30 min before challenge. Survival was monitored.Viability remained constant beyond 72 hours for as long as monitored(two weeks). Experiments involving mice were approved by theinstitutional animal care and use committee.

Statistical Analysis

Survival curves were analyzed using the Kaplan-Meier method, with theLog-Rank test for comparisons.

Structure Models

Cartoon models of protein structure were created in PyMol(<www.pymol.org>).

Example 1

B7-2 Uses its Homodimer Interface to Bind SEB

In the B7-2/CTLA-4 complex (1), the MYPPPY domain as denoted by SEQ IDNO: 59, conserved between CD28 and CTLA-4, engages B7-2 such that theB7-2 dimer interface remains fully accessible. Accordingly, shortpeptides termed herein D-Ala-pB2-4 (having the amino acid sequence(D-A)EKFDSVHSKYM(D-A) denoted by SEQ ID NO: 6) and D-Ala-pB2-6 (havingthe amino acid sequence (D-A)DSDSWTLR(D-A) denoted by SEQ ID NO: 10)overlapping residues in the B7-2 crystallographic homodimer interfacewere synthesized. The peptides are shown in FIG. 1 on the B7-2 dimerinterface region. FIG. 1 shows a variant of SEQ ID NO:13, having theamino acid sequence denoted by SEQ ID NO:50, in which the N-terminal Prowas replaced by Met. As controls, peptides termed herein D-Ala-pB2-2(having the amino acid sequence (D-A)DLPCQFANSQN(D-A) denoted by SEQ IDNO: 2), D-Ala-pB2-3 (having the amino acid sequence (D-A)HHKKPTGMIR(D-A)denoted by SEQ ID NO: 4) and D-Ala-pB2-5 (having the amino acid sequence(D-A)MLKIQAY(D-A) denoted by SEQ ID NO: 8) were synthesized, where thesepeptides fall outside the dimer interface (as shown in FIG. 1 ).

As shown in FIG. 2A, D-Ala-pB2-2, D-Ala-pB2-3 and D-Ala-pB2-5 failed toinhibit SEB-mediated induction of IFN-γ in PBMC whereas D-Ala-pB2-4 andD-Ala-pB2-6 were strongly inhibitory. D-Ala-pB2-4 and D-Ala-pB2-6, butnot the other three peptides, inhibited also induction of IL-2 and TNF-α(FIG. 2B). While at lower concentrations (0.01 μg/ml), pB2-4 and pB2-6inhibited IL-2, IFN-γ and TNF-α induction only partially, together theyshowed pronounced synergistic inhibition (FIG. 2C). By contrast, IL-10induction was resistant to D-Ala-pB2-4 and D-Ala-pB2-6, alone or incombination.

Synergy between D-Ala-pB2-4 and D-Ala-pB2-6 might be derived from thefact that they cover non-overlapping parts of the B7-2 dimer interface(as schematically shown in FIG. 1 ). Therefore their activity was nextcompared to that of a peptide termed herein D-Ala-pB2-7 having the aminoacid sequence (D-A)MGRTSFDSDS(D-A) denoted by SEQ ID NO: 12, whichpartially overlaps D-Ala-pB2-4 and D-Ala-pB2-6, covering a continuoushomodimer interface domain of nine amino acids (FIG. 1 ). As shown inFIG. 2D, pB2-7 inhibited SEB-mediated induction of IL-2, IFN-γ and TNF-αto a comparable extent as that of D-Ala-pB2-4 and D-Ala-pB2-6 incombination and like them, did not inhibit induction of IL-10.

Interestingly, as demonstrated in FIG. 2E, similar results were alsoobtained with streptococcal mitogenic exotoxin Z (SMEZ, FIG. 2E1, FIG.2E2, FIG. 2E3 and FIG. 2E4) which is known to be 40-fold more lethalthan SEB and with toxic shock syndrome toxin-1 (TSST-1, FIG. 2E5, FIG.2E6 and FIG. 2E7), a superantigen having only 6% sequence identity withSEB but structurally conserved in the β-strand(8)/hinge/α-helix(4)domain (12).

The ability of B7-2 dimer interface mimetic peptides to inhibitsignaling for inflammatory cytokine induction by divergent superantigenssuggested that these peptides compete with B7-2 in binding superantigen.Indeed, as shown in FIG. 3A and FIG. 3B, the peptides D-Ala-pB2-4 andD-Ala-pB2-6, respectively, each bound SEB directly, with a K_(D) in themicromolar range (Table 2, below). D-Ala-pB2-4 and D-Ala-pB2-6 similarlybound TSST-1 and SMEZ (FIG. 3C and FIG. 3D for binding TSST-1 and FIG.3E and FIG. 3F for binding SMEZ) with micromolar affinity (Table 2below). Thus, superantigens engage the homodimer interface locatedwithin the V domain of B7-2.

In Table 2 below, purified recombinant superantigens were used andconcentrations of SEB ranged from 0.78 μM in six twofold increments,concentrations of TSST-1 ranged from 0.0625 μM in five twofoldincrements and concentrations of SMEZ ranged from 0.0625 μM in fourtwofold increments.

TABLE 2 Kinetic parameters of Surface Plasmon Resonance analysis % %K_(a) s.e.m. s.e.m. K_(d) s.e.m. s.e.m. KD Ligand Analyte (1/Ms) (K_(a))(K_(a))/k_(a) (1/s) (k_(d)) (K_(d))/k_(d) (μM) X² pB2-4 SEB 413 10.302.50 9.58E−04 4.59E−05 4.80 2.32 1.05 pB2-6 SEB 491 8.79 1.80  1.9E−033.69E−05 1.94 3.90 1.07 pB2-4 TSST-1 4,080 159.00 3.90 2.77E−02 8.00E−052.89 9.80 0.24 pB2-6 TSST-1 2,530 95.40 3.77 3.04E−03 5.92E−05 1.95 1.200.20 pB2-4 SMEZ 3,020 85.00 2.81 2.93E−03 9.32E−05 3.18 7.30 0.40 pB2-6SMEZ 1,590 50.60 3.18 2.86E−03 1.34E−04 4.69 1.79 0.12 K_(a),association rate; k_(d), dissociation rate.

In order to obtain the parameters shown in Table 2 above, purifiedrecombinant superantigens were used. Concentrations of SEB ranged from0.78 μM in six twofold increments; concentrations of TSST-1 ranged from0.0625 μM in five twofold increments; and concentrations of SMEZ rangedfrom 0.0625 μM in four twofold increments. k_(a), association rate;k_(d), dissociation rate. BIAevaluation 3.1 software (BIAcore) was usedto determine KD in the linear ligand concentration range (1:1 Langmuirbinding). Criteria for KD determination for complexes were standarderror as percent of k_(a) and k_(d); λ² values well below 2 show thequality of fit between calculated and observed binding values and attestto the purity of the ligands examined. Fitting residuals were within therange of ±2, validating the goodness of fit.

Example 2

Binding of SEB to B7-2 or CD28: Reciprocal Inhibition by Dimer InterfacePeptides

Next, binding of SEB to a cell population expressing B7-2 was studied.As shown in FIG. 4A, binding of SEB was abrogated by cB7-2 antibody butnot by cCD28, a monoclonal antibody that targets a CD28 dimer interfaceepitope, showing specificity. Interestingly, alone or in combination,the B7-2 dimer interface mimetic peptides D-Ala-pB2-4 and D-Ala-pB2-6effectively blocked binding of SEB to cells expressing B7-2, whereas thepeptide D-Ala-pB2-2, which falls outside the dimer interface, did notinhibit binding (FIG. 4A). The peptide D-Ala-pB2-7 likewise blockedbinding of SEB to cells expressing B7-2, whether alone or in combinationwith D-Ala-pB2-4 (FIG. 4B).

The finding that B7-2 dimer interface mimetic peptides specificallyblock binding of SEB to cell-surface B7-2 shows that the SEB bindingsite in cell-surface B7-2 is the dimer interface and strongly reinforcesthe results showing that these peptides bind superantigens directly andinhibit inflammatory cytokine induction.

Remarkably, D-Ala derivatives of the p2TA and p1TA peptides (having thecore amino acid sequence SPMLVAYD denoted herein by SEQ ID NO: 16 andHVKGKHLCP denoted herein by SEQ ID NO: 17, respectively) derived fromthe CD28 homodimer interface that bind the superantigen directly,inhibiting inflammatory cytokine induction and toxicity (10), equallyinhibited binding of SEB to cells expressing B7-2 (FIG. 4B and FIG. 4C).Because p2TA and p1TA bind SEB in its β-strand(8)/hinge/α-helix(4)domain (10), they compete with B7-2 for its binding site in thesuperantigen.

In a reciprocal experiment, cells were transfected to expresscell-surface CD28. It was previously reported that binding of SEB tosuch cells is abrogated by αCD28, p1TA and p2TA but not by cB7-2 (10).Indeed, the peptides D-Ala-pB2-4, D-Ala-pB2-6 and D-Ala-pB2-7, but notthe control peptide D-Ala-pB2-2, were as capable of blocking binding ofSEB to cell-surface CD28 as CD28 dimer interface mimetics p1TA and p2TA(FIG. 4D).

Thus, the superantigen not only uses its conservedβ-strand(8)/hinge/α-helix(4) domain to bind to either B7-2 or CD28 attheir dimer interface but peptide mimetics of either dimer interfacepossess dual antagonist activity, blocking binding of superantigen toeither receptor in a reciprocal manner.

Example 3

B7-2 Dimer Interface Peptides Protect from SEB Lethality

Next, an accepted model for superantigen lethality,D-galactosamine-sensitized mice was used for demonstrating the effect ofthe peptides D-Ala-pB2-4, D-Ala-pB2-6 and D-Ala-pB2-7 on SEB-challengedmice (10 and 12). As shown in FIG. 5A, D-Ala-pB2-4 and D-Ala-pB2-6blocked superantigen lethality. Whereas only ⅕ of the control micesurvived SEB challenge, ⅘ and 5/5 of the mice that received D-Ala-pB2-4and D-Ala-pB2-6, respectively, at time of exposure to SEB survived (FIG.5A). Notably, the peptide D-Ala-pB2-7 protected when present in only 2-to 3-fold molar excess over SEB (FIG. 5B). This high efficacy supports acritical role for the B7-2 dimer interface in mediating the deleteriousresponse to superantigens.

Example 4

Attenuation of CD28 Signaling by Peptides Derived from the B7-2 DimerInterface

The effect of the peptide D-Ala-pB2-7 was also examined in human PBMCinduced by αCD3, as described above. As shown in FIG. 6A and FIG. 6B,D-Ala-pB2-7 did not inhibit the induction of IFN-γ by αCD3, whether thepeptide was present at 1 μg/ml (FIG. 6A) or a tenfold higherconcentration of 10 μg/ml (FIG. 6B), showing that it does not blocksignaling through the T cell receptor. Moreover, at neitherconcentration, namely 1 or 10 μg/ml did D-Ala-pB2-7 induce a response byitself (FIG. 6A and FIG. 6B, respectively).

However, as shown in FIG. 7A and FIG. 7B, pB2-7 attenuated theexpression of IFN-γ (FIG. 7A) and TNF-α (FIG. 7B) when the expression ofthese cytokines was induced by αCD3 jointly with αCD28. Interestingly, asignificant attenuation of the expression of each of IFN-γ and TNF-α wasobserved at low peptide concentrations of 0.01 and even 0.001 μg/ml,attesting to potent inhibition. Thus, the peptide inhibits signaling foran inflammatory cytokine response when it is transduced through CD28. Asshown in FIG. 8 , inhibition of CD28 signaling was also shown for thecytokines IFN-γ and TNF-α by the peptide D-Ala-pB2-4, which likeD-Ala-pB2-7 is derived from the homodimer interface of B7-2, but not byD-Ala-pB2-2, which falls outside the dimer interface domain.

Example 5

Attenuation of LPS Signaling by the Peptides pB2-4, pB2-6 and pB2-7

Lipopolysaccharide (LPS) is a virulence factor specific forGram-negative bacteria and more generally is a hallmark of Gram-negativeinfection. As shown in FIG. 9A, induction of TNF-α expression in humanPBMC by E. coli LPS was sensitive to attenuation by the B7-2 dimerinterface mimetic peptides D-Ala-pB2-4, D-Ala-pB2-6 as well as by theircombination. The effect of the B7-2 dimer interface mimetic peptidepB2-7 is shown in FIG. 9B.

The greater extent of inhibition demonstrated in FIG. 9A for thecombination of D-Ala-pB2-4 with D-Ala-pB2-6 (versus the effect of eachone of the peptides alone) reflects its greater superantigen antagonistactivity, consistent with the results shown above.

Remarkably, as shown in FIG. 10 , induction of TNF-α expression in humanPBMC induced by E. coli LPS was reduced by each one of the peptidesD-Ala-pB2-4, D-Ala-pB2-6 and D-Ala-pB2-7 even when the peptides werepresent at a concentration of 0.01 μg/ml. Thus, a low concentration ofB7-2 dimer interface mimetic peptide suffices to attenuate LPS-mediatedTNF-α induction.

Example 6

The Potency of B7-2 Dimer Interface Mimetic Peptides

The B7-2 dimer interface has no known role in co-stimulation and isstructurally well separated from the CD28 binding site. Within areceptor homodimer interface, weak, short-range Van der Waalsinteractions coupled with steric fit enable receptor homodimerizationand prevent the generation of heterodimers. Engagement of a superantigenshould displace contacts between the B7-2 monomers, which unlike CD28are not linked through an intermolecular disulfide bond outside thedimer interface and thus require a second-order binding reaction toredimerize. This may explain the ability of D-Ala-pB2-7 to protect micefrom lethal SEB challenge even at low molar ratio to the toxin as shownabove.

B7-2 dimer interface mimetic peptides may bind back into theself-adhesive B7-2 receptor homodimer interface from which they arederived, and thereby attenuate signaling even in the absence of asuperantigen, as shown in FIG. 7 , FIG. 8 and FIG. 9 . Peptide efficacyis enhanced by the lack of an intermolecular disulfide bond between theB7-2 monomers, facilitating their separation by a competing shortpeptide. This may account for the efficacy of B7-2 dimer interfacemimetic peptides against excessive immune stimulation observed in theabsence of a superantigen.

Example 7

Peptides Derived from the B7-1 Homodimer Interface

B7-1 is homologous to B7-2, where both B7-1 and B7-2 act asco-stimulatory ligands expressed on the surface of antigen presentingcells (APCs). FIG. 11 shows the peptides derived from the B7-1 homodimerinterface (MNIWPEYKNRTIFDITNNLSIV, as denoted by SEQ ID NO: 57), namelypB1-4, pB1-6, pB1-7 and pB1-8 denoted by SEQ ID NO: 38, SEQ ID NO: 42,SEQ ID NO: 40 and SEQ ID NO: 48, respectively. FIG. 12 schematicallyshows alignment of segments derived from the dimer interface of bothhuman B7-1 (SEQ ID NO: 57) and human B7-2 (EKFDSVHSKYMGRTSFDSDSWTLR, asdenoted by SEQ ID NO: 58), with conserved residues indicated by boldletters and residues participating in the B7-2 dimer interface indicatedby underlining. FIG. 12 also shows the human B7-2 peptides pB2-4, pB2-7and pB2-6 aligned with the peptides pB1-4, pB1-7 and pB1-6 in part ofthe B7-1 sequence. The effect of the peptides derived from human B7-1dimer interface on cytokine levels as a result of various inductions,for example, superantigen challenge, will be also examined.

Example 8

Model of Polymicrobial Infection: Cecal Ligation and Puncture (CLP)

The murine cecal ligation and puncture (CLP) model is a clinicallyrelevant model to investigate polymicrobial infections and follow theeffects of therapeutic agents on intra-abdominal infections or sepsis.Specific pathogen-free BALB/c mice (8-12 weeks) and CD1 outbred mice(8-12 weeks) are obtained. All animal studies are approved byInstitutional Animal Care and Use Committees (IACUC) before experimentsare initiated.

The animals are anesthetized with inhaled isoflurane (BaxterPharmaceutical Products Inc., Deerfield, Ill.) (induction atconcentration of 4-5%+0.8-1 L/min, maintenance at 1-3%+0.8-1 L/min).

The cecum is exteriorized through a 1.5 cm midline incision and ligatedwith a 5-0 nylon monofilament suture, at 90% of its length just distalto the ileocecal junction. The cecum is then punctured twice using a 23gauge needle along the ante-mesenteric side of the cecum. Patency isassured by expressing a scant amount of laminal contents throughpuncture site. The organ is returned to the abdominal cavity, fascia andskin are closed, and topical lidocaine and bacitracin are applied at thesurgical site. Each animal receives 20 mg/kg intramuscular dose ofmoxifloxacin (representing suboptimal dose of LD₂₅) and 1 mlsubcutaneous bolus of normal saline. The animals are allowed to bere-warmed until fully conscious and are then returned to their cages.

Tested peptides are specifically D-Ala-pB2-7 (as denoted by SEQ ID NO:12), D-Ala-pB2-2 (as denoted by SEQ ID NO:2), D-Ala-pB2-6 (as denoted bySEQ ID NO:10), D-Ala-pB1-4 (as denoted by SEQ ID NO:39), D-Ala-pB1-7 (asdenoted by SEQ ID NO:41), D-Ala-pB1-6 (as denoted by SEQ ID NO:43),D-Ala-pB1-8 (as denoted by SEQ ID NO:49), administered intravenously.

The efficacy of these peptides according to the invention, when givenintravenously (i.v.) is tested and animals are followed daily for atotal of 7 days for overt signs of sepsis and survival. Any moribundanimals (defined as hypothermic <30° C. and unable to maintain normalbody posture) are euthanized and scored as lethally-infected animals. Atthe end of day 7, survivors are euthanized, and are examined forquantitative microbiology of organ tissues (blood, peritoneum, liver,lung, and spleen).

Dose response relationships of a peptide of the invention, whenadministered to animals subjected to CLP is examined. Animals aretreated with different doses of the peptide of the invention, about 2hours after the surgery. Suboptimal dose of moxifloxacin (LD₂₅) may begiven at time 0, to some or all of the animals. Animals are followed upfor several days and survival rates are determined.

The potential effect of the peptides on cytokine and chemokineproduction following CLP is further investigated.

Balb/c mice are subjected to CLP and are treated with a peptideaccording to the invention at about 2 hours post surgery, without anyaddition of antibiotics. Mice (treated and control non-treated, as wellas sham-operated animals, which serve as additional control) areeuthanized at 12 and 24 hours post surgery, and blood is collected inheparinized syringes by cardiac puncture. Plasma is then obtained bycentrifugation and stored at −70° C. until analyzed. Peritoneal fluidsare obtained from mice by lavage, clarified by centrifugation and storedat −70° C. until analyzed. As a representative of Th1 cytokines, thelevels of TNF-α are measured, and as representatives of chemokines thatare associated with pro-inflammatory response, the levels of RANTES andKC are measured in both blood (plasma) and the local infection site(peritoneal fluid) of the peptide-treated animals.

The levels of additional cytokine/chemokine in the peritoneum and bloodfollowing CLP are evaluated. These include TNF-α, IL-6, IL-17A, IL-10,Rantes, MCP-1 and KC in the peritoneal fluid and plasma taken about 12or about 24 hours after CLP.

Animals subjected to CLP exhibit high load of bacteria in the blood andperitoneal fluid. Bacteria usually invade the blood from the peritonealfluid, and are primarily killed by circulating polymorph nuclear cells(PMN) that recognize bacterial elements bound to macrophage surfaces andsecondarily by the resident macrophages themselves. From the bloodbacteria migrate to the liver and spleen (which are the primary sitesfor clearance of bacteria from the systemic circulation), where they arepicked up by resident macrophages. To study the potential effect of thepeptides of the invention on the bacterial load, the dissemination ofbacteria in these tissues/organs is measured. Mice are subjected to CLPand are divided into groups (n=6-8 in each group), that are eithertreated by the peptide about 2 hours post CLP, or injected with PBS andserve as control, or are sham-operated. None of the animals receivesantibiotics. Mice are euthanized after about 12 and about 24 hours fromsurgery, and tissue samples are obtained from the blood, peritonealfluid, liver kidney and spleen of each animal. Levels of bacteria aremeasured by colony counts and compared between the treated and controlgroups.

Keratinocyte chemokine (KC) is an important component responsible forrecruitment and accumulation of polymorph nuclear cells (PMN) intotarget organs that have been implicated as key process in thedevelopment of systemic inflammation during sepsis, leading to organdysfunction. Therefore, the levels of PMN are evaluated in the spleen,liver and kidney of animals post CLP, and are measured by the activityof myeloperoxidase (MPO), which is a key enzyme associated with PMNactivity, serving as an indirect marker for the presence of neutrophils.MPO activity is measured in homogenized tissues at about 12 and about 24hours post CLP. Readout is performed spectrophotometrically at 460 nm,for 10 min, in one minute intervals.

Further support for the reduced levels of PMN in key organs isexemplified by direct counting of PMN in histological slides, obtainedfrom specific tissues of animals post CLP, after immunohistochemicalstaining, for assessment of neutrophil influx. Formalin-fixed paraffinsections obtained from CLP animals at 24 hours post CLP, are stainedwith Naphthol AS-D chloroacetate esterase (leukocyte-specific esterase),counter-stained with Gills hematoxylin solution and coverslipped.Numbers of neutrophils (esterase positively stained cells) present inthe liver sections are randomly screened (5-7 fields/sample)microscopically, at ×400.

To determine if treatment with the peptides of the invention affects theexpression of CD28 on immune effector cells, the peripheral blood cellsand splenocytes are examined about 12 and about 24 hours followingsurgery.

To test the effect of the peptides of the invention on cellproliferation, ex vivo experiments are performed with isolatedsplenocytes taken from sham, CLP mice treated with or without thepeptide, stimulated with anti-CD3 alone or anti-CD3+anti-CD28 antibodiesand cultured for 72 hours. The splenocyte proliferation index isdetermined and compared to cells taken from sham animals.

Increased apoptotic processes in key organs such as kidney, liver andspleen, play a determining pathogenic role in the outcome of sepsis,contributing to organ failure. Therefore, the potential effect oftreatment with the peptides of the invention on renal and spleenapoptosis in animals subjected to CLP is studied. Apoptosis isdetermined in histological slides taken from animals at about 24 hourspost CLP using TUNEL staining. Slides are examined under a fluorescentmicroscope for evidence of apoptosis.

To compare the extent of sepsis-induced apoptosis following CLP betweenpeptide-treated and vehicle-treated mice, isolated splenocytes are alsostained with an early apoptotic marker, Annexin V, combined with cellsurface marker (CD3, CD4, CD8, B220, Gr-1) and analyzed by flowcytometry.

TABLE 3 Sequences referred to herein SEQ ID NO. Sequence DescriptionSEQ ID DLPCQFANSQN pB2-2 NO. 1 SEQ ID (D-A)DLPCQFANSQN(D-A) D-Ala-pB2-2NO. 2 SEQ ID HHKKPTGMIR pB2-3 NO. 3 SEQ ID (D-A)HHKKPTGMIR(D-A)D-Ala-pB2-3 NO. 4 SEQ ID EKFDSVHSKYM pB2-4 NO. 5 SEQ ID(D-A)EKFDSVHSKYM(D-A) D-Ala-pB2-4 NO. 6 SEQ ID MLKIQAY pB2-5 NO. 7SEQ ID (D-A)MLKIQAY(D-A) D-Ala-pB2-5 NO. 8 SEQ ID DSDSWTLR pB2-6 NO. 9SEQ ID (D-A)DSDSWTLR(D-A) D-Ala-pB2-6 NO. 10 SEQ ID MGRTSFDSDS pB2-7NO. 11 SEQ ID (D-A)MGRTSFDSDS(D-A) D-Ala-pB2-7 NO. 12 SEQ IDPLKIQAYFNE TADLPCQFAN Fragment of human NO. 13 SQNQSLSELV VFWQDQENLVB7-2 LNEVYLGKEK FDSVHSKYMG RTSFDSDSWT LRLHNLQIKD KGLYQCIIHH KKPTGMIRIHQMNSELSVLA SEQ ID FCSGVIHVTK EVKEVATLSC B7-1, CD80 antigen NO. 14GHNVSVEELA QTRIYWQKEK (CD28 antigen ligand KMVLTMMSGD MNIWPEYKNR1, B7-1 antigen), TIFDITNNLS IVILALRPSD EGTYECVVLK isoform CRA_aYEKDAFKREH LAEVTLSVKA (Accession No. EAW79564) SEQ ID YNKKKATVQELD p12ANO. 15 SEQ ID SPMLVAYD p2TA NO. 16 SEQ ID HVKGKHLCP p1TA NO. 17 SEQ IDFNETADLP A peptide derived from NO. 18 B7-2 homodimer interface SEQ ID(D-Ala)FNETADLP(D-Ala) A peptide derived from NO. 19 B7-2 homodimerinterface, abutted by D-Ala on both termini SEQ ID NQSLSELVPeptide derived from NO. 20 B7-2 homodimer interface SEQ ID(D-Ala)NQSLSELV(D-Ala) A Peptide derived from NO. 21 B7-2 homodimerinterface, abutted by D-Ala on both termini SEQ ID YLGKEKFDPeptide derived from NO. 22 B7-2 homodimer interface SEQ ID(D-Ala)YLGKEKFD(D-Ala) A Peptide derived from NO. 23 B7-2 homodimerinterface, abutted by D-Ala on both termini SEQ ID TLRLHNLQPeptide derived from NO. 24 B7-2 homodimer interface SEQ ID(D-Ala)TLRLHNLQ(D-Ala) A Peptide derived from NO. 25 B7-2 homodimerinterface, abutted by D-Ala on both termini SEQ ID YMGRTSFDSDPeptide derived from NO. 26 B7-2 homodimer interface SEQID(D-Ala)YMGRTSFDSD(D-Ala) A Peptide derived from NO. 27 B7-2 homodimerinterface, abutted by D-Ala on both termini SEQ ID VKEVATLSPeptide derived from NO. 28 the homodimer interface of B7-1 SEQ ID(D-Ala)VKEVATLS(D-Ala) Peptide derived from NO. 29 B7-1 homodimerinterface, abutted by D-Ala on both termini SEQ ID VEELAQTRPeptide derived from NO. 30 B7-1 homodimer interface SEQ ID(D-Ala)VEELAQTR(D-Ala) Peptide derived from NO. 31 B7-1 homodimerinterface, abutted by D-Ala on both termini SEQ ID MSGDMNIWPeptide derived from NO. 32 B7-1 homodimer interface SEQ ID(D-Ala)MSGDMNIW(D-Ala) Peptide derived from NO. 33 B7-1 homodimerinterface, abutted by D-Ala on both termini SEQ ID SIVILALRPeptide derived from NO. 34 B7-1 homodimer interface SEQ ID(D-Ala)SIVILALR(D-Ala) Peptide derived from NO. 35 B7-1 homodimerinterface, abutted by D-Ala on both termini SEQ ID YKNRTIFDITPeptide derived from NO. 36 B7-1 homodimer interface SEQ ID(D-Ala)YKNRTIFDIT(D-Ala) Peptide derived from NO. 37 B7-1 homodimerinterface, abutted by D-Ala on both termini SEQ ID MNIWPEYKpB1-4, derived from NO. 38 B7-1 homodimer interface SEQ ID(D-Ala)MNIWPEYK(D-Ala) pB1-4, derived from NO. 39 B7-1 homodimerinterface, abutted by D-Ala on both termini SEQ ID KNRTIFDITNpB1-7, derived from NO. 40 B7-1 homodimer interface SEQ ID(D-Ala)KNRTIFDITN(D-Ala) pB1-7, derived from NO. 41 B7-1 homodimerinterface, abutted by D-Ala on both termini SEQ ID DITNNLSIVpB1-6, derived from NO. 42 B7-1 homodimer interface SEQ ID(D-Ala)DITNNLSIV(D-Ala) pB1-6, derived from NO. 43 B7-1 homodimerinterface, abutted by D-Ala on both termini SEQ ID LGKEKFDSVHSKYMGRTSFDSPeptide derived from NO. 44 DSWTLRLHN B7-2 homodimer interface SEQ ID(D-Ala)LGKEKFDSVHSKYMGRTS A Peptide derived from NO. 45FDSDSWTLRLHN(D-Ala) B7-2 homodimer interface, abutted byD-Ala on both termini SEQ ID SGDMNIWPEYKNRTIFDITNNLSIVILAPeptide derived from NO. 46 B7-1 homodimer interface SEQ ID(D-Ala)SGDMNIWPEYKNRTIFDITNN Peptide derived from NO. 47 LSIVILA(D-Ala)B7-1 homodimer interface, abutted by D-Ala on both termini SEQ IDYKNRTIFD Peptide derived from NO. 48 B7-1 homodimer interface pB1-8SEQ ID (D-Ala)YKNRTIFD(D-Ala) Peptide derived from NO. 49 B7-1 homodimerinterface, abutted by D-Ala on both termini D-Ala-pB1-8 SEQ IDMLKIQAYFNE TADLPCQFAN Variant of SEQ ID NO. 50 SQNQSLSELV VFWQDQENLVNO. 13, in which N- LNEVYLGKEK FDSVHSKYMG terminal P is replacedRTSFDSDSWT LRLHNLQIKD by M (underlined) KGLYQCIIHH KKPTGMIRIH QMNSELSVLASEQ IN MNIWPEYKNRTIFDITNNLSIV Fragment of hB7-1; NO. 57see FIGS. 11 and 12 SEQ ID EKFDSVHSKYMGRTSFDSDSWTLRFragment of hB7-2, see NO. 58 FIG. 12 SEQ ID MYPPPY Fragment of humanNO. 59 CD28

The invention claimed is:
 1. An isolated and purified peptide consistingof the amino acid sequence selected from the group consisting of SEQ IDNO:38 (MNIWPEYK, also designated as peptide pB1-4), SEQ ID NO:40(KNRTIFDITN, also designated as peptide pB1-7), SEQ ID NO:42 (DITNNLSIV,also designated as peptide pB1-6), SEQ ID NO:46(SGDMNIWPEYKNRTIFDITNNLSIVILA) and SEQ ID NO:48 (YKNRTIFD, alsodesignated as peptide pB1-8), or a functional derivative of said aminoacid sequence that is any one of: (i) said amino acid sequence that isextended at the N terminus and/or the C terminus thereof: (a) bycysteine or by lauryl cysteine; (b) by an organic moiety that is notnaturally occurring or by a non-naturally occurring amino acid residue;(c) by N-acetyl or lysyl-palmitoyl residue; (d) by hydrophobic aminoacid residue(s) which is/are a naturally occurring or non-naturallyoccurring amino acid residue(s); or (e) by 1 to 4 consecutive amino acidresidues present in immediately adjacent corresponding positions of theamino acid sequence denoted by SEQ ID NO: 14; (ii) a dimer or multimerof said amino acid sequence or any of the functional derivatives of (i);or (iii) a constrained conformation of said amino acid sequence or anyof the functional derivatives of (i) or (ii), wherein the constrainedconformation is formed by an internal bridge or disulfide bridge; andpharmaceutically acceptable salts or esters of any of said peptides andfunctional derivatives thereof.
 2. The isolated and purified peptideaccording to claim 1, wherein said peptide consists of the amino acidsequence of one of SEQ ID NO:38, 40, 42, 46 or 48 extended at its Nterminus and/or at its C terminus by a D-Ala amino acid residue.
 3. Theisolated and purified peptide according to claim 2, being any one of apeptide consisting of the amino acid sequence (D-A)MNIWPEYK(D-A) (SEQ IDNO: 39, also designated as peptide D-Ala-pB1-4), a peptide consisting ofthe amino acid sequence (D-A)KNRTIFDITN(D-A) (SEQ ID NO:41, alsodesignated as peptide D-Ala-pB1-7), a peptide consisting of the aminoacid sequence (D-Ala)DITNNLSIV(D-Ala) (SEQ ID NO:43, also designated aspeptide D-Ala-pB1-6), and a peptide consisting of the amino acidsequence (D-Ala)YKNRTIFD(D-Ala) (SEQ ID NO:49, also designated aspeptide D-Ala-pB1-8), wherein (D-A) in the amino acid sequence isD-alanine.
 4. A pharmaceutical composition comprising as an activeingredient at least one isolated and purified peptide as defined inclaim 1, optionally further comprising a pharmaceutically acceptablecarrier, diluent, adjuvant and/or excipient.
 5. A pharmaceuticalcomposition comprising as an active ingredient at least one isolated andpurified peptide as defined in claim 3, optionally further comprising apharmaceutically acceptable carrier, diluent, adjuvant and/or excipient.